A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode

Cytidine 5′‐triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography‐mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V_max and K_m of CTPS and verified that an inhibition of the enzyme activity was induced after 3‐deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis‐Menten kinetics for lysates of resting cells were K_m=280±310 μmol/L for UTP, V_max=83±20 pmol/min and, for lysates of activated PBMCs, K_m=230±280 μmol/L for UTP, V_max=379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.     

Amperometric Glucose Biosensors Based on Integration of Glucose Oxidase onto Prussian Blue/Carbon Nanotubes Nanocomposite Electrodes

Nanosized Prussian blue (PB) particles were synthesized with a chemical reduction method and then the PB nanoparticles were assembled on the surface of multiwall carbon nanotubes modified glassy carbon electrode (PB/MWNTs/GCE). The results showed that the PB/MWNTs nanocomposite exhibits a remarkably improved catalytic activity towards the reduction of hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the PB/MWNTs platform by an electrochemically polymerized o‐phenylenediamine (OPD) film to construct an amperometric glucose biosensor. The biosensor exhibited a wide linear response up to 8 mM with a low detection limit of 12.7 μM (S/N=3). The Michaelis–Menten constant K_m and the maximum current i_max of the biosensor were 18.0 mM and 4.68 μA, respectively. The selectivity and stability of the biosensor were also investigated.     

Development of Biosensor for Catechol Using Electrosynthesized Poly(3‐methylthiophene) and Incorporation of LAC Simultaneously

Simultaneous electropolymerization of 3‐methylthiophene and incorporation of Laccase (LAC) was carried out in the presence of propylene carbonate as a medium by amperometric method. This enzyme modified electrode was used for the sensing of polyphenol. Catechol is taken as a model compound for the study. UV‐Vis spectral studies suggest no denaturation of LAC in presence of propylene carbonate. The SEM studies reveal the surface morphology and incorporation of LAC in P3MT with agglomerated flaky masses are observed in with and without enzyme micrographs. The cyclic voltammograms were recorded for 0.01 mM catechol on plain glassy carbon, polymer and enzyme incorporated electrodes at pH 6.0 and scan rate 50 mV s^?1. The fabricated electrochemical biosensor was used for the determination of catechol in aqueous solution by Differential Pulse Voltammetry (DPV) technique. The concentration linear range of 8×10^?8 to 1.4×10^?5 M a value of Michealis? Menten constant K_m=7.67 µmol dm^?3 and activation energy is 32.75 kJ mol^?1. It retains 83 % of the original activity after 60 days which is much higher than that of other biosensors. The developed biosensor was used to quantify catechol in the determination in real samples.     

L-Dopa Synthesis on Conducting Polymers

With regards to the synthesis of L-Dopa (l-3,4-dihydroxy phenylalanine) two types of biosensors were designed by immobilizing tyrosinase on conducting polymers; polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT). PPy and PEDOT were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (V_max) and Michaelis Menten constant (K_m) were determined. V_max were found as 0.013 for PPy matrix and 0.041 μ mol/min.electrode for PEDOT matrix. K_m values were determined as 3.7 and 5.2mM for PPy and PEDOT matrices respectively. Calibration curves for enzyme activity vs. substrate concentration were drawn for the range of 0.8 to 2.5 mM L-Tyrosine. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.     

The multiphase kinetics of phosphate uptake by plant roots

The multiphase character of phosphate uptake kinetics was stated in the experiments with intact maize roots depending on different phosphorus concentration in the external solution /from 0.0001 to 50 mM P/. The single phases are characterized by different K_m and V_max values as well as by transition points /T/. The separate phases of the multiphase isotherm of phosphates uptake are distinguished by transition points. T determines the critical concentration of phosphorus as a function of active membrane transport system /i.e. T_1–2=0.39 mM; T_2–3=1.96 mM; T_3–4=10 mM; T_4–5=33 mM/.     

Laccase Production by the Aquatic Ascomycete Phoma sp. UHH 5-1-03 and the White Rot Basidiomycete Pleurotus ostreatus DSM 1833 During Submerged Cultivation on Banana Peels and Enzyme Applicability for the Removal of Endocrine-Disrupting Chemicals

This work aimed to study the production of laccase from Pleurotus ostreatus DSM 1833 and Phoma sp. UHH 5-1-03 using banana peels as alternative carbon source, the subsequent partial purification and characterization of the enzyme, as well the applicability to degrade endocrine disruptors. The laccase stability with pH and temperature, the optimum pH, the K _m and V _max parameters, and the molar mass were determined. Tests were conducted for assessing the ability of degradation of the endocrine disruptors t-nonylphenol, bisphenol A, and 17??-ethinylestradiol. Laccase production of 752 and 1,117?U?L^?1 was obtained for Phoma sp. and P. ostreatus, respectively. Phoma sp. laccase showed higher stability with temperature and pH. The laccase from both species showed higher affinity by syringaldazine. The culture broth with banana peels induced the production of two isoforms of P. ostreatus (58.7 and 21?kDa) and one of Phoma sp. laccase (72?kDa). In the first day of incubation, the concentrations of bisphenol A and 17??-ethinylestradiol were reduced to values close to zero and after 3?days the concentration of t-nonylphenol was reduced in 90% by the P. ostreatus laccase, but there was no reduction in its concentration by the Phoma sp. laccase.     

Purification and Characterization of Laccase Secreted by <Emphasis Type="Italic">L. lividus</Emphasis>

The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K _m, pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 μM, 5.0, and 60 °C, respectively. The K _m values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 μM, respectively.     

d-xylose transport by Candida succiphila and Kluyveromyces marxianus

The kinetics and regulation of d-xylose uptake were investigated in the efficient pentose fermentor Candida succiphila, and in Kluyveromyces marxianus, which assimilate but do not ferment pentose sugars. Active high-affinity (K _m ∼ 3.8 mM; V _max ∼ 15 nmol/[mg·min]) and putative facilitated diffusion low-affinity (K _m ∼ 140 mM; V _max ∼ 130 nmol/[mg·min]) transport activities were found in C. succiphila grown, respectively, on xylose or glucose. K. marxianus showed facilitated diffusion low-affinity (K _m ∼ 103 mM; V _max ∼ 190 nmol/[mg·min]) transport activity when grown on xylose under microaerobic conditions, and both a low-affinity and an active high-affinity (K _m ∼ 0.2 mM; V _max ∼ 10 nmol/[mg·min]) transport activity when grown on xylose under fully aerobic conditions.     

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole,nicotine and DMXAA

A selective and low‐cost CD‐MEKC method under acidic conditions was developed for investigating the N‐oxygenation of tamoxifen (TAM) by flavin‐containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N‐oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β‐CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis–Menten constant (K_m) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V_max) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K_i) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti‐tumour agent, were investigated.     

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K _m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V _max is 99.4 ± 2.85 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent Michaelis constant or K _m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V _max is 102.6 ± 1.73 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent K _m and V _max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min⁻¹ mg⁻¹ protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V _max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.     

Decolorization of Remazol Brilliant Blue R by a Purified Laccase of <Emphasis Type="Italic">Polyporus brumalis</Emphasis>

A white rot basidiomycete Polyporus brumalis has been reported to induce two laccase genes under degradation conditions of dibutylphthalate. When this fungus was grown in a minimal medium, one laccase enzyme was detected by the native polyacrylamide gel electrophoresis. A laccase was purified through ammonium sulfate precipitation and ion exchange chromatography, and the estimated molecular weight was 70 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The K _m value of the enzyme was 685.0 μM, and the V _max was 0.147 ODmin⁻¹ unit⁻¹ for o-tolidine. Purified laccase showed effective decolorization of a dye, Remazol Brilliant Blue R (RBBR), without any laccase mediator. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was directly involved in the decolorization of RBBR.     

ULTRAVIOLET (254-405 nm) ACTION SPECTRUM AND KINETIC STUDIES OF ALANINE UPTAKE IN ESCHERICHIA COLI B/R*

Abstract Uptake of ala in exponentially grown and starved cells of Escherichia coli B/r is inhibited by monochromatic far–UV (254–310 nm) and near UV (310–405 nm) light. The action spectrum for inhibition of ala uptake is similar to that found earlier for gly uptake, showing a maximum at 280 nm and a significant but much lower action throughout the near–UV region. The action spectra suggest that the chromophores for inactivation of ala and gly uptake lie in the carrier proteins and that these proteins are similar. Kinetic studies, in unirradiated bacteria, show that (a) the K_m for ala uptake (11 μM) is about twice that for gly uptake (4.9 μM), (b) the K_m for ala uptake does not change in the presence of gly, although the K_max does decrease, and (c) other amino acids, such as ser and phe, have no effect on the K_m or V_max of the ala uptake system. These data suggest that ala and gly are transported by the same carrier protein, with the binding sites for ala and gly on different subunits.     

Comparative Analysis of the Antiviral Activity of Camel,Bovine, and Human Lactoperoxidases Against Herpes Simplex Virus Type 1

Lactoperoxidase is a milk hemoprotein that acts as a non-immunoglobulin protective protein and shows strong antimicrobial activity. Bovine milk contains about 15 and 7 times higher levels of lactoperoxidase than human colustrum and camel milk, respectively. Human, bovine, and camel lactoperoxidases (hLPO, bLPO, and cLPO, respectively) were purified as homogeneous samples with specific activities of 4.2, 61.3, and 8.7 u/mg, respectively. The optimal working pH was 7.5 (hLPO and bLPO) and 6.5 (cLPO), whereas the optimal working temperature for these proteins was 40 °C. The K _m of hLPO, cLPO, and bLPO were 17, 16, and 19 mM, and their corresponding V _max values were 2, 1.7, and 2.7 μmol/min ml. However, in the presence of H₂O₂, the K _m values were 11 mM for hLPO and cLPO and 20 mM for bLPO, while the corresponding V _max values were 1.17 for hLPO and 1.4 μmol/min ml for cLPO and bLPO. All three proteins were able to inhibit the herpes simplex virus type 1 (HSV-1) in Vero cell line model. The relative antiviral activities were proportional to the protein concentrations. The highest anti-HSV-1 activity was exhibited by bLPO that inhibited the HSV particles at a concentration of 0.5 mg/ml with the relative activity of 100%.

     

Enantioselective oxidation of secondary alcohols at a quinohaemoprotein alcohol dehydrogenase electrode

Quinohaemoprotein alcohol dehydrogenase fromComamonas testosteroni was co-immobilized with a redox polymer (a poly(vinylpyridine) complex functionalized with osmium bis(bipyridine) chloride) on an electrode. The enzyme electrode readily oxidizes primary alcohols and secondary alcohols with maximum current densities varying between 0.43 and 0.98 A m^-2 depending on the substrate and the operation temperature. The affinity of the enzyme for aliphatic alcohols increases with the chain length of the substrate (i.e., 1-pentano1 [K_m = 0.006 mM] is a much better substrate than ethanol [K_m= 2.2 mM]). The same property is observed for secondary alcohols in the series 2-propanol (K_m = 22 mM) to 2-octano1 (K_m = 0.05 mM). The enzyme electrode is enantioselective in the oxidation of secondary alcohols. A strong preference is observed for the S-2-alcohols; the enantioselectivity increases with increasing chain length. The enantiomeric ratio (E) increases from 13 for (R,S)-2-butanol to approximately 80 for (R,S)-2-heptanol and (R,S)-2-octanol. This makes the enzyme electrode, potentially, a powerful tool for the preparation of a large range of alkanones and/or for the (kinetic) resolution of racemic alcohols.     

Amperometric Biosensors Based on Recombinant Bacterial Laccase CotA for Hydroquinone Determination

In this study, stable CotA laccase from Bacillus subtilis 168 was adsorbed on electrode modified with a thiol graphene‐gold nanoparticle (thGP‐AuNPs) nanocomposite film. The novel bacterial laccase biosensor was employed for quantitative detection of hydroquinone (HQ) and the electrochemical properties of this laccase biosensor were investigated. The results indicate that the immobilized CotA shows great oxidation activity towards HQ in the presence of oxygen and the biosensor shows linear electrocatalytic activity in the concentration range from 1.6 to 409.6 μM, with a detection limit of 0.3 μM. Further, the CotA modified electrode, when compared to fungal laccase‐modified biosensors, shows better alkaline stability (retaining approximately 80 % and 70 % of response current at pH 8 and 9, respectively) and reusability (retaining ～87 % of response current after 100 days). The development of this new kind of laccase on a biosensor will offer a novel tool for substance detection applications in hostile environments, especially for industrial pollutants.     

Regression analysis of enzyme reaction. O-methylation of catechol derivatives by catechol-O-methyltransferase

The electronic structures of catechol derivatives, 3,4-dihydroxy-1-substituted benzenes, including epinephrine and norepinephrine, were calculated by means of the CNDO /2 and the MINDO /3 methods. Correlation between the maximum velocities divided by the molecular weight, as between the inverse of Michaelis–Menten constants in the O-methylation reaction catalyzed by catechol-O-methyltransferase and the electronic indices calculated on the basis of the frontier electron theory was found. The correlation coefficient between the observed maximum velocities divided by the molecular weight and the calculated ones was 0.873. The regression analysis showed that the Coulombic forces at the C₃ and C₄ positions and the electron acceptable indices at C₆ and meta-O positions represented the best correlation with the biological activities (V_max and K_m). The electronic indices of the catechol moieties represent the rate-limiting factor of the O-methylation reaction.     

Electrocatalytic Nitrite Reduction to Nitrogen Oxide by a Synthetic Analogue of the Active Site of Cu‐Containing Nitrite Reductase Incorporated in Nafion Film

The electrocatalytic reduction of nitrite to NO by [CuMe₂bpa(H₂O)(ClO₄)]⁺ ( 1 ), which is a model for the active site of copper‐containing nitrite reductase, incorporated in Nafion film was investigated. The Cu complex in the Nafion matrix exhibits an intense band at 267 nm and a broad band around 680 nm, assigned to d–d and ligand field transitions, respectively. The 77‐K EPR spectrum of 1 in the Nafion matrix reveals the typical axial signals (g_//=2.28, g =2.08, A_//=13.3 mT) of a tetragonal Cu²⁺ chromophore. The redox potential, which is related to the Cu⁺/Cu²⁺ couple, was ?146 mV (ΔE=72 mV) at pH 5.5. The redox reaction of 1 in Nafion was not dependent on pH and was a diffusion‐controlled process. The electronic structure and redox properties of 1 in the negatively charged polymer matrix were almost the same as those in aqueous solution. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of 1 in the Nafion matrix. The current increase was dependent on the nitrite concentration and pH in solution. Upon reaching ?400 mV, a linear generation of NO was observed for the 1 /Nafion film coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed with the Michaelis–Menten equation, where V_max=45.1 nM s^?1 and K_m=15.8 mM at pH 5.5. The Cu complex serves the function of both the catalyst and electron transport in the Nafion matrix. The sensitivity of the electrode was estimated to be 3.23 μA mM^?1 in the range of 0.1–0.4 mM nitrite.