THE C-S LYASES OF HIGHER PLANTS. PURIFICATION AND CHARACTERIZATION OF CYSTINE LYASE ISOZYMES FROM TURNIP (BRASSICA RAPA) ROOTS

Cystine lyase, a C-S lyase which utilizes L-cystine and S-alkyl cysteine sulfoxides as substrates, has been shown to exist as two isozymes in several species of Brassica. The isozymes from turnip (Brassica rapa) have been isolated, purified, and their physical and enzymatic properties characterized. Isozyme 1 has a molecular weight of 400, 000 and isozyme 2 one of 440, 000 kD. Both isozymes are glycoproteins possessing 0.3% and 1.1% carbohydrate, respectively, on a weight basis. Both enzymes had optimum activity at pH 8.4. The K_m values for L-cystine were 0.54 mM and 0.42 mM for isozymes 1 and 2, respectively.     

Development of an Electrochemical Sensor for the Determination of Amaranth: a Synthetic Dye in Soft Drinks

The voltammetric behaviour of synthetic food colourant, amaranth, was studied using a multiwalled carbon nanotube (MWCNT) thin film-modified gold electrode in 0.1 M acetate buffer solution of pH 5.0. A well-defined oxidation peak was obtained for amaranth at 0.792 V with the modified electrode. The diffusion controlled oxidation of amaranth at the modified electrode can be attributed to the electrocatalytic nature of MWCNT, since the bare electrode has not produced an electrochemical signal under the same experimental conditions. The operational parameters such as supporting electrolyte, pH of the solution and amount of MWCNT–Nafion suspension were optimised. Under optimum conditions, the oxidation peak current varies linearly with concentration in the range from 1.0?×?10^?5 M to 1.0?×?10^?6 M with a limit of detection at 6.8?×?10^?8 M. The developed sensor has good sensitivity and selectivity towards oxidation of amaranth. The sensor was successfully employed for the determination of amaranth in three different samples of soft drinks, and the results were in good agreement with the standard spectrophotometric method.     

PURIFICATION AND CHARACTERIZATION OF THREEISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purified to homogeneity from tomato (var. S. marzano). The isozymes were separated by affinity chromatography on Heparin-Sepharose column. They exhibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sulfate gel electrophoresis and of 35 kDa in gel-filtration chromatography in native conditions. The isoelectric points of all three isozymes were found to be higher than 9.3. The K_ms calculated for the three isozymes were different toward citrus pectin used as substrate; one had a K_m of 9.7 mM (by expressing the pectin concentration as mmoles/L of methoxy groups) and the other two had similar K_ms of 3.0 and 2.6 mM, respectively. The isozyme having the higher K_m for substrate was inhibited by citrus pectin (which had a degree of methylation of 70%) at concentrations higher than 5 mM, but no inhibition was found using a pectin with a degree of methylation of 30% at concentrations up to 13 mM (i.e. 9 mg/ml) with a K_m of 14.7 mM. Furthermore, this isozyme showed a more broad range of activity in a pH range 5–10 with respect to that exhibited by the other two isozymes. All three isozymes were found to be glycosylated, although to different extents.     

Relative Emulsifying Activity of Bovine Serum Albumin and Casein as Assessed by Three Different Methods

The relative emulsifying activities of bovine serum albumin (BSA) and casein as determined by computerized optical microscopy, electron microscopy and spectroturbidimetry at a protein concentration of 0.135 mM (pH 7) and an oil:water (o:w) ratio of 4:6, was compared. Repeated homogenization led to a more homogeneous distribution of dispersed phase globules. BSA stabilized globules became smaller, e.g. the mean globule diameter determined by the different methods decreased from 3.1 μ at an energy input of 7.6 × 10⁷ J m^?3 to 2.2μ, at 182 × 10⁷ J m^?3. Casein stabilized globules became larger with energy input, e.g. mean d_vs increased from 5.0μ at 7.6 × 10⁷ J m^?3 to 5.8μ. at 182 × 10⁷ J m^?3 indicating structure dependent differences in the emulsifying activity of protein.     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

Electrochemical determination of Sudan I in food products using a carbon nanotube-ionic liquid composite modified electrode

A sensitive and convenient electrochemical method was developed for the determination of Sudan I using a carbon nanotube-ionic liquid composite modified electrode with the enhancement effect of cetyltrimethyl ammonium bromide (CTAB). The modified electrode exhibited an obvious electrocatalytic activity towards the oxidation of Sudan I, and the oxidation peak current significantly increased in the presence of CTAB. The experimental parameters, such as solution pH, concentration of CTAB and accumulation time, were optimised for Sudan I determination. The oxidation peak current showed a linear relationship with the concentration of Sudan I in the range of 3.0 × 10^?8 to 3.1 × 10^?6 mol l^?1, with a detection limit of 8.0 × 10^?9 mol l^?1. The proposed method was successfully applied for the determination of Sudan I in food products of ketchup and chilli sauce.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

Electrochemical Determination of Tert-Butyl Hydroquinone in Edible Oil Samples at Poly (Crystal Violet) Modified Glassy Carbon Electrode

The synthetic phenolic antioxidant tert-butyl hydroquinone (TBHQ) is frequently associated to adverse health effects. A polymerized film of crystal violet (CV) was prepared on the surface of a glass carbon electrode (GCE) by electropolymerization in alkaline solution, and then the modified electrode was successfully used to determine TBHQ. This electrode was characterized by scanning electron microscopy and electrochemical impedance spectra. The voltammetric behavior of TBHQ over an extended pH range using cyclic voltammetry at poly (crystal violet) modified glassy carbon electrode (PCV/GCE) was also studied. The resulting electrode exhibited excellent electrocatalytic activity towards the oxidation of TBHQ, and this was confirmed by the observed increased redox peak currents and shifted potentials. The electrochemical sensor can be applied to the quantification of TBHQ with a linear range covering 5?×?10^?7–1?×?10^?4 mol?·?L^?1 (with a correlation coefficient of 0.9969) and the limit detection was 3?×?10^?8 mol?·?L^?1(S/N?=?3). The recovery was between 97.1 and 102 % in edible oil samples. The electrochemical sensor method was also compared with a HPLC method, which proves its capability in commercial market surveillance.     

Osmotic Dehydration of Nectarines: Influence of the Operating Conditions and Determination of the Effective Diffusion Coefficients

The aim of the present work is to study the kinetics of osmotic dehydration of Caldesi nectarines (Prunus persica var. nectarina) evaluating the effect of osmotic solution concentration, type of solute, temperature, fruit/solute ratio and process time on moisture content, water loss, soluble solids content and solids gain. The process analysis was carried out experimentally and numerically through the mathematical modelling of mass transfer. Hypertonic solutions of glucose syrup and sorbitol (40 and 60 % w/w) were used for dehydration, during 2 h of process at temperatures of 25 and 40 °C, with fruit/osmotic agent ratio of 1:4 and 1:10. Water loss and solids gain showed significant differences depending on the type and concentration of the osmotic agent, process time and fruit/solution ratio. The concentration interacted significantly with all variables; in addition, there was an interaction between the type of osmotic agent and the relationship between fruit and the osmotic agent. The effective diffusion coefficients were obtained from the analytical solution of Fick’s second law applied to flat-plate geometry and by solving the mass transfer microscopic balances by finite element method, taking into account the real geometry of the nectarine pieces. The values obtained from Fick’s law varied between 1.27?×?10^?10 and 1.37?×?10^?08?m²?s^?1 for water and from 1.14?×?10^?10 to 1.08?×?10^?08?m²?s^?1 for soluble solids, while the values calculated by finite elements method ranges were between 0.70?×?10^?09 and 4.80?×?10^?09?m²?s^?1 for water and between 0.26?×?10^?09 and 1.70?×?10^?09?m²?s^?1 for soluble solids. The diffusion coefficients values obtained from the numerical solution are consistent with those published in literature.     

Effect of Temperature and Moisture Content on the Kinetics of Trypsin Inhibitor Activity, Protein in vitro Digestibility and Nitrogen Solubility of Cowpea Flour

Samples of finely ground cowpea flour containing 7.5%, 19.4% and 25.5% moisture were heated in sealed tubes at 100° 125° and 150°C for periods of 0.5 to 120 min. First order rate constants for losses of trypsin inhibitor activity and nitrogen solubility ranged from 1 × 10^?2 to 18 min^?1 and from 4 × 10^?2 to 8 min^?1 respectively. In vitro protein digestibility (IVD) increased, then decreased with heating as described by sequential first order kinetics. Rate constants for increase of IVD varied from 0.13 to 12 min^?1, while for decrease in IVD the range was 5 × 10^?5 to 3 × 10^?2 min^?1. Activation energies and relationships between In k and water activity were computed.     

Regulation of Volatile Fatty Acid Uptake by Mitochondrial Acyl CoA Synthetases of Bovine Liver,

Mitochondria of bovine liver contain acyl CoA synthetases necessary for the uptake of propionate, butyrate, and valerate whereas acetate is bound only weakly. Purification of these enzymes separated a distinct propionyl CoA synthetase highly specific for propionate and acrylate and a butyrate-activating fraction with broad substrate specificity for short and medium chain fatty acids. Evidence from kinetic studies and sucrose density centrifugation suggested that this latter fraction was composed of two enzymes, a butyryl CoA synthetase and a valeryl CoA synthetase. The apparent molecular weights of the propionyl, butyryl, and valeryl CoA synthetases were 72,000, 67,000, and 65,000. The Michaelis-Menten constants of propionyl CoA synthetase for propionate, adenosine 5’-triphosphate, and coenzyme A were 1.3 × 10^?3M, 1.3 × 10^?3M, and 6.3 × 10^?4M. Enzyme activity is regulated by the concentration of propionate in portal blood. Relative to propionyl, butyryl, or valeryl CoA synthetases little acetyl CoA synthetase could be demonstrated.In ruminants hepatic metabolism is such that use of acetate as an energy source is minimum. This ensures that an alternative energy source to glucose, as acetate units, will reach the extrahepatic tissues. Separation of a distinct propionyl CoA synthetase regulated by the concentration of propionate in portal blood is significant because a primary role of ruminant liver is to synthesize glucose from ruminally derived propionate.     

Thermal Inactivation Kinetics of Wheat Germ Lipoxygenase

Thermal inactivation curves for wheat germ lipoxygenase (LPO) in partially purified and crude extracts were determined in capillary tubes at 60–68°C. The biphasic curves fitted a two-fraction first order model suggesting the presence of 2 groups of isozymes. At 60°C, the inactivation rate constants were 9.112 × 10^?-⁵ set^?1 and 9.174 × 10^?-⁶ set^?1 respectively, for thermolabile phase I and thermostable phase II in the partially purified extract, a difference of one order of magnitude. For the temperature change from 60 to 68°C, the rate constants increased by three orders of magnitude, implying a very high sensitivity (for LPO inactivation in partially purified extract ΔH^?= 646261 J.mole^?1, ΔS^?= 1619 J.mole^?1.K^?1 for phase I, ΔH^?= 546099 J.mole-^l, ΔS^?= 1298 J.mole^?1.K-1^′ for phase II) to heat by both phases, although phase I was clearly the least stable.     

Thermal Properties of Kerstingiella geocarpa Seeds as Influenced by Moisture Content

The specific heat capacity, thermal conductivity and thermal diffusivity of Kerstingiella geocarpa seeds were determined as a function of moisture content. The initial moisture content of the seeds determined using the ASAE standard test was 10.0 % (d.b). The specific heat capacity of Kerstingiella geocarpa seed increased from 155.83 to 204.45 Jkg^?1k?1, as the moisture content increased from 10 to 30 % (d.b). The thermal conductivity of the seed increased from 5.13 × 10^?2 to 4.87 × 10^?1 Wm^?1k^?1, as the moisture content increased. The thermal diffusivity of the seed increased from 2.35 × 10^?4 to 3.66 × 10^?3 m²s^?1, as the moisture content increased. These values indicate the ability of the Kerstingiella geocarpa seed to retain heat when processed. The regression models that could be used to adequately express the relationships existing between the thermal properties of the Kerstingiella geocarpa seed and moisture content were established.     

Inhibitory effects of selected pesticides on peroxidases purified by affinity chromatography

The objective of this study was to determine the in vitro inhibition effects of seven commonly used pesticides including 2,4-d-acid dimethylamine, fenoxaprop-p-ethyl, glyphosate isopropylamine, haloxyfop-p-methyl, cypermethrin, λ-cyhalothrin, and dichlorvos on the peroxidase purified from turnip (Brassica rapa L.) and black radish (Raphanus sativus L.) using 4-amino benzohydrazide affinity column chromatography. The purification factors for the turnip and black radish peroxidases were found to be 263.29-fold (with a yield of 12.89%) and 36.20-fold (with a yield of 6.90%), respectively. Among these compounds, λ-cyhalothrin showed the strongest inhibitory effect against turnip peroxidase (K_i: 1.23 × 10^?2 ± 0.21 × 10^?2 mM) as noncompetitive inhibition. On the other hand, cypermethrin demonstrated the highest inhibition effect against black radish peroxidase (K_i: 2.14 × 10^?2 ± 0.08 × 10^?2 mM) as competitive inhibition.     

A maltose,L-rhamnose sensor based on porous Cu foam and electrochemical amperometric i-t scanning method

A maltose, L-rhamnose sensor based on porous Cu foam and electrochemical techniques was investigated in this paper. Cu foam material was prepared and characterized by scanning electron microscopy (SEM). The electro-oxidation reaction process of sweeteners occurred on Cu foam electrode was evaluated by cyclic voltammetry (CV) scanning. At an applied potential of 0.5 V, the linear range for maltose is 0.18–3.47 mM with sensitivity of 1.0492 mA cm^?2 mM^?1. The limit of detection (LOD) was 15.86 μM (S/N?=?3). The linear range for maltose is 0.18–3.47 mM with sensitivity of 0.6881 mA cm^?2 mM^?1. The LOD was 24.18 μM (S/N?=?3). Compared with Cu sheet electrode, Cu foam electrode showed higher current response towards maltose and L-rhamnose, leading to enhanced electrocatalytic activity, higher sensitivity, and lower LOD. Sweetener qualitative discrimination was carried out by stochastic resonance (SR) signal-to-noise ratio (SNR) spectrum eigen peak located noise intensities.     

Radionuclide concentration in tea, cabbage, orange, kiwi and soil and lifetime cancer risk due to gamma radioactivity in Rize, Turkey

BACKGROUND: In this study, the activity concentrations of ²³²Th, ²³⁸U, ⁴⁰K and ¹³⁷Cs were measured in tea, cabbage, orange, kiwi and soil samples collected from different stations using gamma spectrometry with a high‐purity germanium detector. RESULTS: The average activity concentrations of ²³²Th, ²³⁸U, ⁴⁰K and ¹³⁷Cs were found to be 8.2 ± 1.8, 17.3 ± 3.3, 465.8 ± 11.8 and 20.9 ± 3.8 Bq kg^?1 in food samples, and 72.4 ± 9.8, 51.1 ± 8.3, 229.3 ± 14.7 and 312.9 ± 11.5 Bq kg^?1 in farm soils, respectively. The internal effective dose to individuals and excess lifetime cancer risk from the consumption of the food type radioactivity ranged between 11.7 and 53.6 µSv y^?1 and between 0.05 × 10^?3 and 0.24 × 10^?3, respectively. The annual external gamma effective dose and excess lifetime cancer risk in the farms due to soil radioactivity ranged between 94.1 and 139.8 µSv y^?1 and between 0.43 × 10^?3 and 0.64 × 10^?3, respectively. The mean transfer factors of ²³²Th, ²³⁸U, ⁴⁰K and ¹³⁷Cs, from the soil to vegetables and fruit were 0.57, 0.32, 2.12 and 0.04, respectively. CONCLUSION: Annual effective gamma doses were found to be higher than the world's average in soil samples. The excess lifetime cancer risks were only found higher than the world's average in soil samples. Copyright © 2011 Society of Chemical Industry     

A new potassium tetrabromoaurate (III)-luminol chemiluminescence system for the determination of folic acid in milk powder

Abstract: A new chemiluminescence (CL) system based on the CL‐emitting reaction between potassium tetrabromoaurate (III) [Au(III)] and luminol in alkaline medium is described in this paper. On the basis of this study, folic acid (FA) could dramatically enhance CL intensities, and incorporated with flow injection (FI) and solid‐phase extraction (SPE), the novel CL system has been applied for the determination of FA in infant formula milk powder. Under optimum conditions, the CL intensities were linearly related to the concentration of FA in the range of 8.0 × 10^?8 to 4.0 × 10^?5 g/L with a correlation coefficient of 0.999, and the detection limit was 2.0 × 10^?8 g/L. The relative standard deviation was 3.5% for 1.0 × 10^?6g/L FA. The optimal conditions for the detection of FA were evaluated, and the interferences from some common inorganic ions and a couple of relevant organic compounds were also investigated. Practical Application: The new Au(III)–luminol chemiluminescent system is proposed for the determination of FA; it will be an adequate technique for the analysis of low FA concentrations in pharmaceutical preparations and food samples, and it will have potential analytical applications for other substances.     

α-, γ- and δ-Tocopherol Effects on Chlorophyll Photosensitized Oxidation of Soybean Oil

The effects of 0, 1.0, 2.0 and 4.0 (x 10^?3 M) α-, γ- or δ-tocopherol on chlorophyll b photosensitized oxidation of soybean oil in methylene chloride were studied by peroxide values and headspace oxygen. As concentrations of tocopherols increased, peroxide values decreased and headspace oxygen increased (P < 0.05). At 1.0 × 10^?3 M, α-tocopherol showed highest antioxidant effect, γ-tocopherol second and then δ-tocopherol. α- and -γ-Tocopherols had similar effects and δ-tocopherol had lower effect at 2.0 × 10^?3 M (P < 0.05). However, the three tocopherols were not different (P > 0.05) at 4.0 × 10^?3 M. α-Tocopherol quenched singlet oxygen to reduce the photosensitized oxidation of oil. The quenching rate constants of α-tocopherol were 2.7 × 10⁷M^?1sec^?1 by peroxide value and 2.6 × 10⁷ M^?1sec^?1 by headspace oxygen.     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.