Immobilization of -glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm⁻² and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10⁻³ mol l⁻¹ and that of free enzyme was 4.8 × 10⁻² mol l⁻¹. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl⁻¹ within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl⁻¹. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Dictyostelium discoideum chemotaxis: Threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10⁻³ nM/μm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10⁻¹ nM/μm. In very steep gradients, above 10 nM/μm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Succinylated bovine heart mitochondrial cytochromec oxidase

Cytochromec oxidase was prepared by sequential extraction of bovine heart muscle submitochondrial particles with sodium deoxycholate, followed by fractional precipitation with ammonium sulfate and chromatography on Sephadex G-75. The resulting preparation had typical absorption spectra, an activity of 1.28 sec^–1 (mg protein)^–1 (3 ml)^–1 in deoxycholate or 4.13 sec^–1 (mg protein)^–1 (3 ml)^–1 in 0.5% Tween 80, and a minimum molecular weight of 120,000 daltons as calculated from the heme content and the total protein. Amino acid analyses of nine preparations yielded a molecular weight per heme of 86,500 daltons. The net charge was calculated to be +8.7 at pH 7.0. Succinylation of cytochromec oxidase in the presence of 500 molar excess of succinic anhydride produced a soluble preparation having a negative charge at neutral pH. The modified enzyme was highly autoxidizable and had little or no activity toward ferrocytochromec as a substrate. Its averageS _20,w was 5.8 and its apparentD was 4.0 × 10^–7 cm² sec^–1, from which a molecular weight of 126,000 daltons was calculated. This size of enzyme is considered to be that of the monomer, because the value is practically the same as the minimum molecular weight reported herein, and since it is approximately onehalf the value obtained in our laboratory (and in others) for the unmodified enzyme.     

Aerotaxis and chemotaxis ofAzospirillum brasilense: A note

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3–4×10⁴, after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10⁴–1.1×10⁵ cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.     

Extracellular Glucose Oxidase of Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1 using ultrafiltration membranes was developed. Two samples of the enzyme with a specific activity of 914–956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16°C was 2.74 × 10^–6 s^–1. This constant decreased significantly as the pH of the medium increased (4.0–10.0). The temperature optimum for glucose oxidase–catalyzed -D-glucose oxidation was in the range 30–65°C. At temperatures below 30°C, the activation energy for -D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase–catalyzed -D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and<Emphasis Type="Italic"> Arthrobacter globiformis</Emphasis>: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ_amr-Cu(II)TPQ_sq-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O₂ for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K_d values for Cu(II)-CN^– and Cu(I)-CN^–, as well as the K_i for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN^– complexation of Cu(I).Abbreviations AGAO Arthrobacter globiformis amine oxidase - APAO Arthrobacter P1 amine oxidase - APT attached proton test - BPAO bovine plasma amine oxidase - CuAO quinone-copper containing amine oxidase - LTQ lysyl tyrosylquinone - MAO monoamine oxidase - PKAO porcine kidney amine oxidase - PPAO porcine plasma amine oxidase - PSAO pea seedling amine oxidase - TPQ 2,4,5-trihydroxyphenylalaninequinone - TPQ_amr TPQ aminoresorcinol - TPQ_imq TPQ iminoquinone - TPQ_ox TPQ oxidized - TPQ_sq TPQ semiquinone - WT wild-typeE.M. Shepard and G.A. Juda contributed equally to this workThis revised version was published online in February 2004: Hansenula polymorpha was not italicised at the end of the Introduction, Equation 3 appeared twice, and the resolution of Scheme 3 was insufficient.An erratum to this article can be found at     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

Role of Chemotaxis in the Ecology of Denitrifiers

A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO₃⁻ and NO₂⁻ by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10⁻³ M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10⁻² M), tryptone, and a soil extract, but not to NH₄⁺, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO₃⁻, NO₂⁻, or a soil extract, and the chemoattraction to NO₃⁻ occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO₃⁻ was usually observed when the cells were grown aerobically with 10 mM NO₃⁻ in the growth medium. Capillary tubes containing 10⁻3 M NO₃⁻ submerged into soil-water mixtures elicited a significant chemotactic response to NO₃⁻ by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO₃⁻ and NO₂⁻, and which may facilitate the survival of naturally occurring populations of some denitrifiers.     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K _d 6.7 × 10^–7 M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavinbinding site of the enzyme, apo-L-aspartate oxidase has been reacted with N⁶-(6-carboxyhexyl)-FAD Succinimidoester. The structural characterization of the resulting N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/ monomer. Residue Lys38 was identified as the target of the covalent modification. N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.     

Lysyl oxidase dependent synthesis of a collagen cross-link containing histidine

To date, collagen appears unique among proteins in that it contains histidine in certain of its cross-links. Synthesis of histidine containing collagen cross-links was studied in vitro with lathyritic L-¹⁴C histidine or L-¹⁴C lysine labelled bone collagen fibrils and purified lysyl oxidase. Synthesis of the tetrafunctional cross-link, dehydrohistidinohydroxymerodesmosine occurred with lysyl oxidase and was inhibited by β-aminopropionitrile. Synthesis began after a lag period of sixteen hours and then proceeded linearly for four days. These data indicate that enzyme dependent synthesis of dehydrohistidinohydroxymerodesmosine occurs in vitro in a biochemically defined system. Biosynthesis in vivo might occur under similar conditions.     

Effects of butyltins and inorganic tin on chemotaxis of aquatic bacteria

Summary Tributyltin (TBT) and its degradation products dibutyltin (DBT), monobutyltin (MBT) and Sn^IV were toxic toPseudomonas fluorescens SHC-6 andSerratia sp. Gil-1 with EC₅₀ values in the range of 10^–3 to 10^–4M. These four compounds were negative chemotactic agents forP. fluorescens, and the butyltins were negatively chemotactic forSerratia sp. at concentrations over four orders of magnitude lower than the EC₅₀ values.l-Aspartate was a positive chemotactic agent for both organisms. TBT, DBT and MBT negated the effect ofl-aspartate onP. fluorescens but not onSerratia sp. Thus, TBT has the potential to affect microbial populations at concentrations much lower than those which prevent growth, and degradation of TBT does not always detoxify it. SnCl₄ was less toxic than TBT or DBT to these organisms and it was not chemotactic forSerratia sp. Gil-1. Tributylamine and tributylphosphate were less than 1/10th as toxic as TBT and they did not have a chemotactic effect on either organism at concentrations at which TBT had a significant effect. Therefore, both the Sn-and butyl-moieties contribute to the toxic and chemotactic properties of TBT.     

Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis

A new NADH oxidase, useful for the regeneration of NAD⁺, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg^–1. After purification by four chromatographic steps, an activity of 116 U mg^–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K _m value for NADH was 24 M.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

Polyamine catabolism in higher plants: Characterization of pyrroline dehydrogenase

Both mono-and dicotyledonous species catabolize putrescine to -aminobutyric acid (GABA), but by two different pathways. GABA is the major labeled product in pea shoots and oat leaves fed with a 2–4 h pulse of [1,4-¹⁴C]-putrescine (Put) or [1,4-tetramethylene-¹⁴C]-spermidine (Spd), respectively. In the presence of 1–10 M gabaculine, a specific inhibitor of GABA: pyruvate-transaminase, the label appearing in GABA increases 2 to 7-fold, which indicates that the transamination reaction is a major fate of GABA formed from Put or Spd in vivo. The conversions to GABA were demonstrated in vitro in coupled assays involving diamine oxidase from pea or polyamine oxidase from oat, and pyrroline dehydrogenase (PYRR-DH). The latter enzyme from either pea or oat is strictly NAD-dependent and is specific for pyrroline. The optimal temperature (40–45°C) and pH (7.5–8.0) are similar to those of bacterial PYRR-DH. In all cases the enzyme was inhibited by the NAD analogs thionicotinamide and aminopyridine dinucleotide (0.1–1.0 mM). In addition to pea and oat, PYRR-DH was also detected in corn, barley, soybean and broadbean. Di- and polyamine oxidase are released by enzymes which degrade the cell wall, while PYRR-DH remains associated with the protoplast.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.