Obeying Michaelis-Menten Kinetics Research Articles

Comparative detoxification of Remazol Rrilliant Blue R by free and immobilized laccase of Oudemansiella canarii

A laccase from Oudemansiella canarii was immobilized using the crosslinked enzyme aggregate (CLEA) methodology and applied in the degradation of the anthraquinonic dye Remazol Brilliant Blue R (RBBR). The immobilized laccase was superior to the free laccase in both thermal and storage stabilities. Both immobilized and free laccase decolourized 100 mg/L RBBR within 24 h at 30 °C and pH 5.0, but the former was still efficient in degrading the dye after at least 6 cycles. The relationship between the decolorization rate and the RBBR concentration obeyed Michaelis–Menten kinetics, with KM of 0.126 ± 0.044 mmol/L and Vmax of 1.412 ± 0.295 µmol/min for free laccase and KM of 0.159 ± 0.050 mmol/L and Vmax of 1.214 ± 0.242 µmol/min for immobilized laccase. Fourier transform infra-red spectroscopy (FTIR) and mass spectrometry allowed to conclude that the O. canarii laccase acts not only on the chromophore group of the dye, but that it also cleaves other covalent bonds, causing an effective fragmentation of the molecule. The Microtox assay detected a significant diminution in toxicity, a finding corroborated by the phytotoxicity test performed with lettuce seeds. Our results indicate that the immobilized laccase from O. canarii could be useful in biological strategies aiming at degrading unwanted dyes in the environment.

Enzymatic degradation and detoxification of azo dye Congo red by a new laccase from Oudemansiella canarii

A single laccase with molecular weight of 41 kDa was produced by the white-rot fungus Oudemansiella canarii cultured on solid state fermentation using a mixture of sugarcane bagasse-wheat bran as substrate. The enzyme (5 U) was able to decolourize 80% of 50 mg/L Congo red within 24 h at 30 °C and pH 5.5. The relationship between the decolorization rate and dye concentration obeyed Michaelis–Menten kinetics, with KM and Vmax values of 46.180 ± 6.245 µM and 1.840 ± 0.101 µmol/min, respectively. Fourier transform infrared spectroscopy (FTIR) and mass spectrometry allowed to conclude that the laccase acts not only on the dye chromophore group, but also that it cleaves different covalent bonds, causing an effective fragmentation of the molecule. The action of the laccase caused a significant reduction in toxicity, as indicated by the Microtox test. In conclusion, O. canarii laccase could be useful in future biological strategies aiming at degrading azo dyes.

Dopamine binding directly up-regulates (Na+, K+)-ATPase activity in the gills of the freshwater shrimp Macrobrachium amazonicum

We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100 mmol L−1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VM = 190.5 ± 15.7 nmol Pi min−1 mg−1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.

A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110kDa and hydrolyzes ATP with V M=146.8±6.3nmol Pi min-1mg protein-1 and K M=0.05±0.003mmolL-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V M=139.4±6.9nmol Pi min-1mgprotein-1, K M=4.50±0.22mmolL-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M=136.8±6.5nmol Pi min-1 mg protein-1, K 0.5=0.27±0.04mmol L-1), K+ (V M=140.2±7.0nmol Pi min-1 mg protein-1, K 0.5=0.17±0.008mmol L-1), and NH4+ (V M=149.1±7.4nmol Pi min-1 mg protein-1, K 0.5=0.60±0.03mmol L-1) shows cooperative kinetics. Ouabain (K I=52.0±2.6µmol L-1) and orthovanadate (K I=1.0±0.05µmol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

Low salinity-induced alterations in epithelial ultrastructure, Na+/K+-ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab, Clibanarius vittatus (Anomura, Diogenidae).

Fresh caught Clibanarius vittatus [SW, 31‰ salinity (S)] were acclimated to a dilute medium (15‰ S) for 10 days, employing silver staining to locate gill ion transporting tissue, immunofluorescence to localize the Na+/K+-ATPase α-subunit in the lamellae, and electron microscopy to portray ultrastructural changes in the gill epithelia. Na+/K+-ATPase activity was characterized kinetically in a gill microsomal fraction, including synergistic stimulation by NH4+ plus K+. Silver staining revealed that all 26 phyllobranchiate arthro- and pleurobranchiae participate in ion transport. Na+/K+-ATPase α-subunit staining was weak in SW crabs and distributed exclusively and irregularly within the intralamellar septal cells, particularly at the septal-pillar cell body junctions, and septal cell cytoplasm facing the hemolymph space. In 15‰ S crabs, α-subunit localization was intense, occupying the entire thickened septum. Pillar cells and flanges did not stain. Mitochondria and membrane foldings increased in the pillar cell flanges and intralamellar septal cells, greatly amplifying surface area. Only a single ATP binding site (VM=130.8±10.5nmolmin-1mgprotein-1; K0.5=55.3±1.7μmoll-1) obeying Michaelis-Menten kinetics was disclosed. Na+/K+-ATPase activity was modulated by Mg2+, Na+, and NH4+, exhibiting site-site interactions; K+ modulation showed Michaelis-Menten kinetics. K+ plus NH4+ synergistically stimulated activity≈1.7-fold. Ouabain inhibited total ATPase activity by≈70% (KI=220-300μmoll-1), revealing phosphohydrolytic activities other than the Na+/K+-ATPase. Despite ample phylogenetic separation, the phyllobranchiate lamellae of the Anomura and Caridea share many ultrastructural features, that is, an intralamellar septum and opposed abutting pillar cells, similar Na+/K+-ATPase distribution, and comparable kinetic characteristics. These findings suggest either convergent evolution at the structural and biochemical levels, or preservation of traits present in a remote common ancestor.

Noise slows the rate of Michaelis–Menten reactions

Microscopic randomness and the small volumes of living cells combine to generate random fluctuations in molecule concentrations called “noise”. Here I investigate the effect of noise on biochemical reactions obeying Michaelis–Menten kinetics, concluding that substrate noise causes these reactions to slow. I derive a general expression for the time evolution of the joint probability density of chemical species in arbitrarily connected networks of non-linear chemical reactions in small volumes. This equation is a generalization of the chemical master equation (CME), a common tool for investigating stochastic chemical kinetics, extended to reaction networks occurring in small volumes, such as living cells. I apply this equation to a generalized Michaelis-Menten reaction in an open system, deriving the following general result: 〈p〉≤p¯ and 〈s〉≥s¯, where s¯ and p¯ denote the deterministic steady-state concentration of reactant and product species, respectively, and 〈s〉 and 〈p〉 denote the steady-state ensemble average over independent realizations of a stochastic reaction. Under biologically realistic conditions, namely when substrate is degraded or diluted by cell division, 〈p〉≤p¯. Consequently, noise slows the rate of in vivo Michaelis–Menten reactions. These predictions are validated by extensive stochastic simulations using Gillespie's exact stochastic simulation algorithm. I specify the conditions under which these effects occur and when they vanish, therefore reconciling discrepancies among previous theoretical investigations of stochastic biochemical reactions. Stochastic slowdown of reaction flux caused by molecular noise in living cells may have functional consequences, which the present theory may be used to quantify.

Purification, characterization and thermostability improvement of xylanase from Bacillus amyloliquefaciens and its application in pre-bleaching of kraft pulp

Xylanases have important industrial applications but are most extensively utilized in the pulp and paper industry as a pre-bleaching agent. We characterized a xylanase from Bacillus amyloliquefaciens strain SK-3 and studied it for kraft pulp bleaching. The purified enzyme had a molecular weight of ~50 kDa with optimal activity at pH 9.0 and 50 °C. The enzyme showed good activity retention (85%) after 2 h incubation at 50 °C and pH 9.0. This enzyme obeyed Michaelis–Menten kinetics with regard to beechwood xylan with Km and Vmax values of 5.6 mg/ml, 433 μM/min/mg proteins, respectively. The enzyme activity was stimulated by Mn2+, Ca2+ and Fe2+ metal ions. Further, it also showed good tolerance to phenolics (2 mM) in the presence of syringic acid (no loss), cinnamic acid (97%), benzoic acid (94%) and phenol (97%) activity retention. The thermostability of xylanase was increased by 6.5-fold in presence of sorbitol (0.75 M). Further, pulp treated with 20U/g of xylanase (20IU/g) alone and with sorbitol (0.75M) reduced kappa number by 18.3 and 23.8%, respectively after 3 h reaction. In summary, presence of xylanase shows good pulp-bleaching activity, good tolerance to phenolics, lignin and metal ions and is amenable to thermostability improvement by addition of polyols. The SEM image showed significant changes on the surface of xylanase-treated pulp fiber as a result of xylan hydrolysis.

Multiple alternative substrate kinetics

The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the “specificity constant” (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis–Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

A kinetic characterization of (Na+, K+)-ATPase activity in the gills of the pelagic seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae).

We characterize the kinetic properties of a gill (Na(+), K(+))-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na(+), K(+))-ATPase activity, but also containing mitochondrial F0F1- and Na(+)- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈ 110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na(+), K(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with VM = 109.5 ± 3.2 nmol Pi min(-1) mg(-1) and KM = 0.03 ± 0.003 mmol L(-1). Mg(2+) (VM = 109.8 ± 2.1 nmol Pi min(-1 )mg(-1), K0.5 = 0.60 ± 0.03 mmol L(-1)), Na(+) (VM = 117.6 ± 3.5 nmol Pi min(-1 ) mg(-1), K0.5 = 5.36 ± 0.14 mmol L(-1)), K(+) (VM = 112.9 ± 1.4 nmol Pi min(-1 )mg(-1), K0.5 = 1.32 ± 0.08 mmol L(-1)), and NH4 (+) (VM = 200.8 ± 7.1 nmol Pi min(-1 )mg(-1), K0.5 = 2.70 ± 0.04 mmol L(-1)) stimulated (Na(+), K(+))-ATPase activity following site-site interactions. K(+) plus NH4 (+) does not synergistically stimulate (Na(+), K(+))-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K(+) binding that can be occupied by NH4 (+), stimulating the enzyme. Ouabain (KI = 84.0 ± 2.1 µmol L(-1)) and orthovanadate (KI = 0.157 ± 0.001 µmol L(-1)) inhibited total ATPase activity by ≈ 50 and ≈ 44 %, respectively. Ouabain inhibition increases ≈ 80 % in the presence of NH4 (+) with a threefold lower KI, suggesting that NH4 (+) is likely transported as a K(+) congener.

Characteristics of lipase in dormant seeds catalysed hydrolysis of olive oil in SDS‐olive oil reversed microemulsions

The activity of lipase‐catalysed hydrolysis of olive oil in SDS‐1‐butanol‐olive oil‐water microemulsions was investigated. The kinetics of lipases in reverse microemulsions and water were also compared. The influences of the characteristic parameters of these systems, such as pH, temperature (T), molar ratio (ωo) of water to SDS, the concentrations of enzyme and substrate, on the activity of an enzyme were examined. According to the optimum conditions the production and activity were measured at pH = 4, T = 40 °C, ωo = 12. The activation energy of the reaction was calculated from the Arrhenius plot. It was found that the hydrolysis reaction obeyed Michaelis–Menten kinetics and the apparent Michaelis Km,a and the apparent maximal reaction rate Vmax were determined. The effect of microemulsion's surfactant (SDS) on enzymatic activity was also discussed.

Human CTP:phosphoethanolamine cytidylyltransferase: Enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis–Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.

Iron colloids reduce the bioavailability of phosphorus to the green alga Raphidocelis subcapitata

Phosphorus (P) is a limiting nutrient in many aquatic systems. The bioavailability of P in natural waters strongly depends on its speciation. In this study, structural properties of iron colloids were determined and related to their effect on P sorption and P bioavailability. The freshwater green alga Raphidocelis subcapitata was exposed to media spiked with radiolabelled 33PO4, and the uptake of 33P was monitored for 1 h. The media contained various concentrations of synthetic iron colloids with a size between 10 kDa and 0.45 μm. The iron colloids were stabilised by natural organic matter. EXAFS spectroscopy showed that these colloids predominantly consisted of ferrihydrite with small amounts of organically complexed Fe. In colloid-free treatments, the P uptake flux by the algae obeyed Michaelis–Menten kinetics. In the presence of iron colloids at 9 or 90 μM Fe, corresponding to molar P:Fe ratios between 0.02 and 0.17, the truly dissolved P (<10 kDa) was between 4 and 60% of the total dissolved P (<0.45 μm). These colloids reduced the P uptake flux by R. subcapitata compared to colloid-free treatments at the same total dissolved P concentration. However, the P uptake flux from colloid containing solutions equalled that from colloid-free ones when expressed as truly dissolved P. This demonstrates that colloidal P did not contribute to the P uptake flux. It is concluded that, on the short term, phosphate adsorbed to ferrihydrite colloids is not available to the green alga R. subcapitata.

Production, purification, and characterization of a highly thermostable and humic acid biodegrading peroxidase from a decolorizing Streptomyces albidoflavus strain TN644 isolated from a Tunisian off-shore oil field

This study reports on the production, purification, and characterization of a new extracellular humic acid peroxidase (termed HaP4, following the names given to HaP1-3) from decolorizing actinomycetes strain isolated from Tunisian off-shore oil field and assigned as Streptomyces albidoflavus strain TN644 based on physiological and biochemical characteristics and 16S rRNA gene sequencing. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that HaP4 is a monomer with a molecular mass of 50,011.17 Da and a Reinheitzahl (RZ) value of 1.55. Its N-terminal 27 amino-acid sequence showed high homology with those of Streptomyces humic acid peroxidases. HaP4 showed optimal activity at pH 4 and 70 °C using 2,4-dichlorophenol (2,4-DCP) as a substrate. The enzyme was inhibited by sodium azide, mercury, cadmium, cyanide, and l-cystine, suggesting that heme components were present in its tertiary structure. The findings revealed that it was a heme protein catalyzing the oxidation of various substrates in the presence of hydrogen peroxide (H2O2). HaP4 obeyed Michaelis–Menten kinetics, and its catalytic efficiency (kcat/Km) was higher than that of the commercial enzyme horseradish peroxidase (HRP).

Propensity approach to nonequilibrium thermodynamics of a chemical reaction network: Controlling single E-coli β-galactosidase enzyme catalysis through the elementary reaction steps

In this work, we develop an approach to nonequilibrium thermodynamics of an open chemical reaction network in terms of the elementary reaction propensities. The method is akin to the microscopic formulation of the dissipation function in terms of the Kullback-Leibler distance of phase space trajectories in Hamiltonian system. The formalism is applied to a single oligomeric enzyme kinetics at chemiostatic condition that leads the reaction system to a nonequilibrium steady state, characterized by a positive total entropy production rate. Analytical expressions are derived, relating the individual reaction contributions towards the total entropy production rate with experimentally measurable reaction velocity. Taking a real case of Escherichia coli β-galactosidase enzyme obeying Michaelis-Menten kinetics, we thoroughly analyze the temporal as well as the steady state behavior of various thermodynamic quantities for each elementary reaction. This gives a useful insight in the relative magnitudes of various energy terms and the dissipated heat to sustain a steady state of the reaction system operating far-from-equilibrium. It is also observed that, the reaction is entropy-driven at low substrate concentration and becomes energy-driven as the substrate concentration rises.

Effect of organic solvents on peroxidases from rice and horseradish: Prospects for enzyme based applications

A feasibility test for rice peroxidase (RP) enzyme as a substitute for horseradish peroxidase (HRP) was carried out. The activity of HRP was maximum at 30°C with pH 6.0–7.0. The purified rice peroxidase showed optimum activity at 30°C with pH 7–8 and was thermostable till 68°C, which is higher than the temperature reported for HRP. RP obeyed Michaelis–Menten kinetics. With increasing substrate concentrations, RP and HRP had Vmax as 8.23μMmin−1 and 4.21μMmin−1 and Km as 5.585 and 3.662mM, respectively. In 10% 1,4-dioxane and ethanol, RP exhibited 2 and 1.3 times higher activity, respectively than HRP. Shelf life studies show RP to be significantly stable till 60h in 20% 1,4-dioxane and till 12h in ethanol. The activity of RP/HRP increased gradually with 0%–40% ethanol or 0%–30% 1,4-dioxane till 20h with a sharp decline thereafter. The stability of HRP and RP reduced with increasing storage period. Enzyme efficiencies compared as Vm/Km showed water miscible organic solvents, viz.1,4-dioxane and ethanol, to exhibit a regular decrease in Vm/Km with increase in organic solvent concentration whereas, a reverse trend was observed with water–immiscible solvent like chloroform. The relative activity of RP and HRP enzymes upon immobilization on poly-5-carboxy-indole shows increasing enzyme activity with time and with guaiacol/dopamine hydrochloride as substrates. Immobilized RP had a better relative activity with dopamine as substrate than immobilized HRP, whereas with guaiacol both RP and HRP had a comparable activity upon immobilization. Results suggest rice peroxidase to be a cheaper and convenient enzyme system for immobilization using organic solvents. The high thermal stability, more stability in organic solvents and longer shelf life of RP over the immobilizing matrix suggest conducting polyindole having carboxyl functional groups to be a suitable matrix for the covalent entrapment of rice peroxidase through amide linkage. Good sensitivity and fast response to dopamine makes RP a suitable enzyme system for monitoring dopamine levels in aqueous medium.

Stable isotope liquid chromatography–tandem mass spectrometry assay for fatty acid amide hydrolase activity

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d4-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d4-EA) and the internal standard 13C2-EA. Selected reaction monitoring of m/z 66→m/z 48 (d4-EA) and m/z 64→m/z 46 (13C2-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d4-AEA hydrolysis obeyed Michaelis–Menten kinetics (KM=12.3μM, Vmax=27.6nmol/minmg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC50=24.3nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.

Leishmania donovani encodes a functional enzyme involved in vitamin c biosynthesis: Arabino-1,4-lactone oxidase

Plants and most animals can synthesize ascorbate (vitamin C) for their own requirements, but humans have lost this ability during evolution. The last step in the biosynthesis of l-ascorbic acid involves the conversion of an aldonolactone substrate to ascorbate (or analogues), reactions catalyzed by a family of flavoprotein aldonolactone oxidase/dehydrogenases. We report cloning, molecular characterization, localization and functional importance of arabinonolactone oxidase (LdALO), an enzyme from L. donovani, a protozoan parasite that causes visceral leishmaniasis. L. donovani arabinonolactone oxidase gene is 1509-bp and encodes a putative 502-amino acid protein with a molecular mass of 57-kDa. A 57-kDa protein was obtained by heterologous expression of LdALO in Escherichia coli. Recombinant arabinonolactone oxidase (LdALO) obeys Michaelis–Menten kinetics utilizing d-arabinono-γ-lactone as a substrate, a property characteristic of the yeast enzyme. Activity towards the mammalian substrate, l-gulono-γ-lactone, could not be detected. The inhibitor study profile suggested the essentiality of cysteine residues for the activity of this enzyme. LdALO displayed glycosomal localization as in other kinetoplastids. Overexpression of LdALO in L. donovani resulted in better ability of survival of the parasite within the host in comparison to the vector transfectants. d-Arabinono-γ-lactone oxidase required for synthesizing ascorbate in Leishmania could be considered as a therapeutically exploitable target.

Guidelines for optimal design of coflow enzyme microreactors

Enzyme microreactors are used as research tools because of many advantages including low reagent consumption and the general notion that mass transfer restrictions are reduced. The use of microchannels can indeed shorten the characteristic mass transfer time but may also affect the productivity of the microreactor. To what extent mass transfer restrictions affect the reaction rate and the productivity is determined by parameters such as the enzyme properties, operating conditions, and dimensions of the microreactor. This article provides the correlations between these parameters for coflow enzyme microreactors obeying Michaelis–Menten kinetics. These correlations outline the design space based on reduced mass transfer restrictions and maximum productivity, respectively. The methodology that yields the design space provides a generic hands-on approach to optimally design coflow enzyme microreactors. We conclude that effectiveness is an important parameter for designing microreactors, but – depending on the reactor configuration – does not necessarily lead to optimized throughput. We demonstrate design windows to illustrate this effect and how to take advantage of it.

The influence of composition and structure of water-in-oil microemulsions on activities of Iraqi Turnip peroxidase

The activity of the enzyme Iraqi Turnip peroxidase (ITP) is studied in a reverse microemulsion composed of chloroform, aqueous buffer, sodium dodecylsulfate (SDS) and alcohols of the homologous series 1-propanol to 1-hexanol through the measurements of absorbancy of the product of oxidation at the wavelength of 470nm in the course of reactions. The ITP catalyzed reaction is the oxidation of guaiacol by hydrogen peroxide. Maximum enzyme activity was obtained at ω0 (molar ratio of water to surfactant)=8. It was found that the oxidation reaction obeyed Michaelis–Menten kinetics in the investigated concentration rang (0.08–0.8mM) of the substrate, and the Michaelis constant Km and maximal reaction rate Vm were determined. The enzyme inhibition caused by the alcohols in microemulsions is a consequence of both the solubility of the alcohols in the buffer and the flexibility of the interfacial film.

Peroxidase Activity of DNA Aptamer–Pt Complexes Prepared with Cisplatin

DNA aptamers carrying Pt nanoparticles prepared with cisplatin showed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. Optimal preparation conditions of DNA–Pt complex prepared with cisplatin were investigated on the synthesis at pH 7–11, a reaction time of 1–18 h and 90°C. The enzymatic reaction of DNA–Pt complex obeyed Michaelis–Menten kinetics. K M for the DNA–Pt complex was found to be of the same order as K M for hemin and hemin–DNA complex, but one order of magnitude higher than that of horseradish peroxidase. A sandwich type of DNA enzyme-linked aptamer assay (DLAA) using DNA–Pt complex successively detected target protein of thrombin. DLAA using DNA–Pt complex fractioned by ultrafiltration membranes having a molecular weight cut-off of 30 000 and 300 000 showed 1.9-times higher sensitivity than DLAA using DNA–Pt complex without fraction. The DNA–Pt complex having specific size was effective for the sensitive detection of thrombin in DLAA.