Michaelis-Menten Kinetic Parameters Research Articles

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

HKUST-1 catalyzed efficient in situ regeneration of NAD+ for dehydrogenase mediated oxidation

The combination of enzymatic and chemical reactions to achieve co-operative chemo-enzymatic processes has presented many challenges for decades. In this work, alcohol dehydrogenase (ADH) was encapsulated into metal-organic framework HKUST-1 crystals through an in situ approach to construct a synergistic chemo- and bio-catalysis system, where ADH was for the conversion of benzyl alcohol to benzaldehyde, and HKUST-1 was in charge for the regeneration of β-nicotinamide adenine dinucleotide (NAD+, the oxidized cofactor). It was found that HKUST-1 was a versatile catalyst for efficient oxidation of NADH in different buffers. The Michaelis-Menten kinetic parameters of immobilized ADH exhibited an increased KM (approximately 1.3-fold) and a decreased Vmax (approximately 4.7-fold) compared to free ADH. Due to the protection of HKUST-1, the immobilized enzyme showed a better resistance against urea and organic solvents compared with free enzyme, and still maintained 93% of its original activity after 6 cycles. The immobilized enzyme was successfully applied to the enzymatic transformation of benzyl alcohol to benzaldehyde in aqueous media at room temperature. Owing to the oxidized cofactor regeneration by HKUST-1, ADH@HKUST-1 composites achieved a benzaldehyde yield of 28.5%, which was approximately 4.3-fold higher than that of the individual bio-catalysis. This concept of combining chemo- and bio-catalysis may provide a novel and versatile technique for applications in biochemical catalysis, biosensors and drug delivery.

Performance evaluation of a bottom liner incorporated up-flow anaerobic sludge blanket reactor start-up for food waste

A bottom biofilter liner incorporated up-flow anaerobic sludge blanket reactor was designed, developed, and evaluated for food wastes over 47 days. It was operated as a semi-batch setup with intermittent up-flow circulation (very infrequent) to simulate the start-up with phosphate, Ca(OH)2, and fresh cow dung. Volatile solids (VS), volatile suspended solids (VSS), salinity, pH, biochemical oxygen demand, chemical oxygen demand, and phosphate were monitored. An analytical procedure was developed by summing up total VSS over the lapse time from t = 0 for the operational period which gave overall reaction rate of v and the total cumulative values of VS as substrate S for applying Michaelis–Menten kinetics for optimizing and predicting reactor performance. At ideal operating conditions, pH ranged from 5.69 at the bottom to 7.9 at top stratum. The salinity at the bottom stratum reduced from 8.8 to 0.5 ‰, indicating excellent performance of the liner. There are two ways of analysing phase behaviour and applying Lineweaver Burk plot, one gave Michaelis–Menten kinetic parameters of vm=44.25 g/L/day and Km=76.02 g/L for 4th phase and vm=86.96 g/L/day and Km=66.26 g/L for 5th phase. Non-competitive inhibitions can be identified between these phases, thus best to arrest recirculation after 21 days and continuous feeding from then on.

LeNRT1.1 Improves Nitrate Uptake in Grafted Tomato Plants under High Nitrogen Demand.

Grafting has become a common practice among tomato growers to obtain vigorous plants. These plants present a substantial increase in nitrogen (N) uptake from the root zone. However, the mechanisms involved in this higher uptake capacity have not been investigated. To elucidate whether the increase in N uptake in grafted tomato plants under high N demand conditions is related to the functioning of low- (high capacity) or high-affinity (low capacity) root plasma membrane transporters, a series of experiments were conducted. Plants grafted onto a vigorous rootstock, as well as ungrafted and homograft plants, were exposed to two radiation levels (400 and 800 µmol m−2 s−1). We assessed root plasma membrane nitrate transporters (LeNRT1.1, LeNRT1.2, LeNRT2.1, LeNRT2.2 and LeNRT2.3) expression, Michaelis‒Menten kinetics parameters (Vmax and Km), root and leaf nitrate reductase activity, and root respiration rates. The majority of nitrate uptake is mediated by LeNRT1.1 and LeNRT1.2 in grafted and ungrafted plants. Under high N demand conditions, vigorous rootstocks show similar levels of expression for LeNRT1.1 and LeNRT1.2, whereas ungrafted plants present a higher expression of LeNRT1.2. No differences in the uptake capacity (evaluated as Vmax), root respiration rates, or root nitrate assimilation capacity were found among treatments.

Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450.

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

Microwave Irradiation-Assisted Chitosan Hydrolysis Using Cellulase Enzyme

The influence of microwave irradiation on the chitosan hydrolysis catalyzed by cellulase enzyme was studied. The hydrolyzed chitosan was characterized by measuring its viscosity and reducing sugar. Further, it was also characterized by Fourier-Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), and Scanning Electron Microscope (SEM). The classical Michaelis-Menten kinetic parameters were measured by analyzing the amount of reducing sugars. The results were compared with the hydrolysis by using conventional shaker incubator. The hydrolysis reaction time needed to obtain similar reducing sugar yield was significantly lower for microwave irradiation than shaker incubator. On the other hand, the reduction rate of the relative viscosity was significantly higher for the hydrolysis of chitosan using shaker incubator. A significant difference in chemical structure was observed between hydrolysis using microwave irradiation and shaker incubator. Overall, the result showed that the hydrolysis behavior of chitosan using microwave irradiation is significantly different with using shaker incubator.

Sunflower oil transesterification with methanol using immobilized lipase enzymes.

The transesterification of sunflower oil with methanol, using immobilized lipase enzymes as catalysts, was studied. The process was carried out in a semi-continuous mode. Temperature (30-50°C), methanol flow (0.024-0.04ml/min), kind of enzyme (Lipozyme 62350, Lipozyme TL-IM, Novozym 435 and Pseudomonas cepacia Sol-Gel-AK) and enzyme concentrations (1.25-10% by weight) were the operating variables. The final product was characterized by the EN 14214 standard. All the parameters, except for cold filter plugging point, were similar to a diesel fuel. For low methanol flows, reaction rate was proportional to methanol concentration. High flows caused catalyst deactivation. Novozyme 435, Lipozyme TL-IM and Lipozyme 62350 showed similar maximum reaction rates, but Novozyme 435 was more resistant to deactivation. Pseudomonas cepacia hardly obtained 1% conversion. The catalyst concentration, up to 2.5%, had a positive effect on the reaction rate and conversion. The optimum temperature was 40°C. The initial reaction rate was in line with the Arrhenius law, up to 50°C. By differential and integral methods, the Michaelis-Menten, competitive inhibition and ping-pong bi-bi kinetic parameters were determined. The transesterification of sunflower oil in a semi-continuous regime of alcohol improved the results, compared to the discontinuous regime, and those were similar to the obtained ones in a discontinuous regime with step-by-step methanol addition. The lipase that showed the best yield and higher resistance to deactivation was Novozym 435. The kinetic models that forecast the deactivation of lipases by an inhibitor described the experimental behavior properly.

Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography-mass spectrometry. Michaelis-Menten kinetic parameters were determined. For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Capillary Electrophoresis-Based Enzyme Assay for Nicotinamide N-Methyltransferase

A capillary electrophoresis method was developed for the determination of the activity of nicotinamide N-methyltransferase. Separation of the substrate nicotinamide and the product N-methylnicotinamide as well as the cofactor S-(5′-adenosyl)-l-methionine and its metabolite S-(5′-adenosyl)-l-homocysteine were achieved in a 50/60.5 cm fused-silica capillary using 100 mM sodium phosphate buffer, pH 3.0 as electrolyte and an applied voltage of 25 kV. Analyte detection was carried out at 193 nm. Using lidocaine as internal standard the method was validated for nicotinamide and N-methylnicotinamide with regard to range, limit of detection, limit of quantitation, repeatability, intermediate precision and accuracy. The assay was applied to the determination of the Michaelis–Menten kinetic parameters of the enzyme.

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.

A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity

Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (Kmand Vmax) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants.

Receptor protein of Lysinibacillus sphaericus mosquito-larvicidal toxin displays amylomaltase activity

The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23–560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM (Km) and 1.9 s−1 (kcat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 (Kd, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated Kd values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature (Tm) as observed by thermofluor-shift assay is 51.5 °C and Ca2+ provides structural stability to the protein.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

Kinetic of carbonic anhydrase immobilized onto amberlite xad 7 and it application in sequestration of co2 into caco3 precipitate

Carbonic Anhydrase (CA) was immobilized onto Amberlite XAD 7 and was used in carbon dioxide sequestration purposes. The catalytic activities for free and immobilized CA were estimated by using para-Nitrophenyl Acetate (p-NPA) as the substrate in Tris-buffer containing 10% of acetonitrile. Lineweaver-Burk plot was employed to estimate the Michaelis-Menten kinetic parameters for both free and immobilized CA. Km value of free and immobilized CA were 2.92 mM and 5.7 mM respectively. Meanwhile the Vmax value of free and immobilized CA were 5.95 μmoles/min/ml and 2.67 μmoles/min/ml respectively. The kinetic value obtained in the present study shown that the immobilized CA has high affinity for its substrate. On the other hand, activity and stability study at various pH and temperature indicates that the optimum pH for free CA was found to be at pH 9 while for immobilized CA was at pH 10. For optimum temperature, a free CA was performed optimally at temperature 25°C and immobilized CA was working effectively at temperature 50°C. The immobilized CA onto Amberlite was tested in the CO2 sequestration process and the formation of the white CaCO3 precipitate was observed during the process. CaCO3 powder obtained during the process was validated with the XRD analysis. The finding indicated that immobilized CA onto Amberlite XAD7 retained it enzymatic activity and stability and thus perform well in the CO2 sequestration which gave the white CaCO3 precipitate.

The investigation of α-amylase inhibitory activity of selected Pinto bean peptides via preclinical study using AR42J cell

The current work investigated the α-amylase inhibitory activity of five selected Pinto bean peptides (PBPs) using AR42J cell. The objectives were: (a) to prove the potential of PBPs as α-amylase inhibitor in living cells, and (b) to investigate the inhibition mode of PBPs. Results showed that PBP9 obtained the lowest IC50 value of 0.31mM followed by PBP7 (5.92mM), PBP1 (6.08mM), PBP6 (6.14mM) and PBP3 (6.64mM). In the MTT assay, all PBP showed a dose-dependent trend, whereby lower concentration of PBP demonstrated a higher cell viability. By using PBP concentrations of 0.33–8.3mM, Michaelis-Menten kinetic parameters and Lineweaver-Burk plots revealed that Km and Vmax values were ranged from 38.52–794.38mM and 0.72–1.15mM/min, respectively, resulting in uncompetitive (PBP3, PBP6 and PBP7) and unusual (PBP1 and PBP9) α-amylase inhibition modes. It was suggested that PBP could be considered as new agents for the diabetes treatment.

Identification of the upstream 4-chlorophenol biodegradation pathway using a recombinant monooxygenase from Arthrobacter chlorophenolicus A6

This study aimed to clarify the initial 4-chlorophenol (4-CP) biodegradation pathway promoted by a two-component flavin-diffusible monooxygenase (TC-FDM) consisting of CphC-I and CphB contained in Arthrobacter chlorophenolicus A6 and the decomposition function of CphC-I. The TC-FDM genes were cloned from A. chlorophenolicus A6, and the corresponding enzymes were overexpressed. Since CphB was expressed in an insoluble form, Fre, a flavin reductase obtained from Escherichia coli, was used. These enzymes were purified using Ni2+-NTA resin. It was confirmed that TC-FDM catalyzes the oxidation of 4-CP and the sequential conversion of 4-CP to benzoquinone (BQN)→hydroquinone (HQN)→HQL. This indicated that CphC-I exhibits substrate specificity for 4-CP, BQN, and HQN. The activity of CphC-I for 4-CP was 63.22U/mg-protein, and the Michaelis-Menten kinetic parameters were vmax=0.21mM/min, KM=0.19mM, and kcat/KM=0.04mM−1min−1. These results would be useful for the development of a novel biochemical treatment technology for 4-CP and phenolic hydrocarbons.

Oxidative biodegradation of 4-chlorophenol by using recombinant monooxygenase cloned and overexpressed from Arthrobacter chlorophenolicus A6

In this study, cphC-I and cphB, encoding a putative two-component flavin-diffusible monooxygenase (TC-FDM) complex, were cloned from Arthrobacter chlorophenolicus A6. The corresponding enzymes were overexpressed to assess the feasibility of their utilization for the oxidative decomposition of 4-chlorophenol (4-CP). Soluble CphC-I was produced at a high level (∼50%), and subsequently purified. Since CphB was expressed in an insoluble form, a flavin reductase, Fre, cloned from Escherichia coli was used as an alternative reductase. CphC-I utilized cofactor FADH2, which was reduced by Fre for the hydroxylation of 4-CP. This recombinant enzyme complex exhibited a higher specific activity for the oxidation of 4-CP (45.34U/mg-protein) than that exhibited by CphC-I contained in cells (0.18U/mg-protein). The Michaelis-Menten kinetic parameters were determined as: vmax=223.3μM·min−1, KM=249.4μM, and kcat/KM=0.052min−1·μM−1. These results could be useful for the development of a new biochemical remediation technique based on enzymatic agents catalyzing the degradation of phenolic contaminants.

Effectiveness of Vegetated Drainage Ditches for Domestic Sewage Effluent Mitigation.

Plant species have an important role in eco-ditches; however, the Michaelis-Menten kinetic parameters of nutrient uptake, growth rate and purification efficiency of ditch plants and their influences on domestic sewage treatment efficiency are still unclear. Growth rates of all nine species, but especially Lemna gibba, Cladophora and Myriophyllum verticillatum were best in undiluted domestic sewage as opposed to a mixture of domestic sewage. Performance of species to accumulate nutrients was not only species-specific, but was also affected by both sewage treatments. Removal efficiency of nutrients was dependent on both plant species and treatment. Uptake kinetic parameters were significantly affected by both nutrient form and plant species. The maximum uptake rate (Vmax) of NH4-N was higher than NO3-N. Similarly, Km values for NH4-N were greater than NO3-N. These results could be used to identify plants for sewage treatment efficiency and enhance water quality in eco-ditch treatment systems.

Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity

This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H2O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (KM; Vmax) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars.

Purification and characterization of manganese peroxidases from native and mutant Trametes versicolor IBL-04

Extracellular manganese peroxidases (MnPs) produced by native and mutant strains of Trametes versicolor IBL-04 (EB-60, EMS-90) were purified by ammonium sulphate precipitation and dialysis, followed by ion-exchange and gel-permeation chromatography. The purified enzymes elucidated a single band in the 43-kDa region on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum pH and temperature of the purified enzymes were found to be 5.0 and 40 °C, respectively. Mutant strain MnPs exhibited a broader active pH range and higher thermal stability than native MnP. Purified MnPs from selected mutants showed almost identical properties to native MnP in electrophoresis, steady-state kinetics, and metal ion and endocrine-disrupting compound (EDC) degradation efficiency. Although the fastest reaction rates occurred with Mn2+, MnPs displayed the highest affinity for ABTS, methoxyhydroquinone, 4-aminophenol and reactive dyes. MnP activity was significantly enhanced by Mn2+ and Cu2+, and inhibited in the presence of Zn2+, Fe2+, ethylenediaminetetraacetic acid and cysteine to various extents, with Hg2+ as the most potent inhibitory agent. MnPs from all sources efficiently catalyzed the degradation of the EDCs, nonylphenol and triclosan, removing over 80% after 3 h of treatment, which was further increased up to 90% in the presence of MnP-mediator system. The properties of T. versicolor MnPs, such as high pH and thermal stability, as well as unique Michaelis-Menten kinetic parameters and high EDC elimination efficiency, render them promising candidates for industrial exploitation.