Comparison of detection methods for liquid chromatographic determination of 3-nitro-l-tyrosine

A liquid chromatographic method has been developed for the determination of 3-nitro-l-tyrosine. Different detection methods, including UV, oxidative and redox electrochemistry, and postcolumn photolysis followed by electrochemical detection, have been optimized and compared in terms of analysis time, detection limit and dynamic range. It was demonstrated that liquid chromatography with postcolumn photolysis followed by electrochemical detection is the most effective method, with an analysis time of 5 min, detection limit of 0.01 pmol, and a linear dynamic range from 2 nM to 100 μM.     

Effect of nitrogen source concentration on curdlan production by Agrobacterium sp. ATCC 31749 grown on prairie cordgrass hydrolysates

The effect of nitrogen source concentration on the production of the polysaccharide curdlan by the bacterium Agrobacterium sp. ATCC 31749 from hydrolysates of prairie cordgrass was examined. The highest curdlan concentrations were produced by ATCC 31749 when grown on a medium containing a solids-only hydrolysate and the nitrogen source ammonium phosphate (2.2 mM) or on a medium containing a complete hydrolysate and 3.3 mM ammonium phosphate. The latter medium sustained a higher level of bacterial curdlan production than the former medium after 144 hr. Biomass production by ATCC 31749 was highest after 144 hr when grown on a medium containing a solids-only hydrolysate and 2.2 or 8.7 mM ammonium phosphate. On the medium containing the complete hydrolysate, biomass production by ATCC 31749 was highest after 144 hr when 3.3 mM ammonium phosphate was present. Bacterial biomass production after 144 hr was greater on the complete hydrolysate medium compared to the solids-only hydrolysate medium. Curdlan yield produced by ATCC 31749 after 144 hr from the complete hydrolysate medium containing 3.3 mM ammonium phosphate was higher than from the solids-only hydrolysate medium containing 2.2 mM ammonium phosphate.     

Effect of different acclimation methods on the performance of microbial fuel cells using phenol as substrate

Single-chamber microbial fuel cells (MFCs) with air-cathode were constructed. MFCs were fed different feedstocks during their inoculation, their role on phenol degradation and MFC performance were investigated. The results showed that the MFC inoculated using glucose exhibited the highest power density (31.3 mW m^?2) when phenol was used as the sole substrate for MFC. The corresponding biodegradation kinetic constant was obtained at 0.035 h^?1, at an initial phenol concentration of 600 mg L^?1. Moreover, the phenol degradation rates in this MFC with closed circuit were 9.8–16.5 % higher than those in MFC with opened circuit. The cyclic voltammograms revealed a different electrochemical activity of the anode biofilms in the MFC, and this led to differences in performance of the MFCs with phenol as sole substrate. These results demonstrated that phenol degradation and power production are affected by current generation and type of acclimation.     

Cross-linked enzyme crystals of organophosphate hydrolase for electrochemical detection of organophosphorus compounds

Cross-linked enzyme crystals of organophosphate hydrolase (CLEC-OPH) prepared from crude recombinant E. coli cell lysate was used for the development of an electrochemical biosensor for the detection of organophosphate pesticides. CLEC-OPH showed an increased V _max of 0.721 U mg protein⁻¹ and a slightly lower K _m of 0.083 mM on paraoxon compared to the crude enzyme, resulting in an improved catalytic efficiency (k _cat/K _m = 4.17 × 10⁵ M⁻¹ min⁻¹) with a remarkable increase on thermostability. An amperometric biosensor was constructed based on glutaraldehyde and albumin cross-linkage of CLEC-OPH with carbon nanotubes. The sensor exhibited greater sensitivity and operational stability with a lower limit of detection when compared with a sensor using an equivalent loading of crude OPH in a non-crystal form. The application of crude enzyme-based CLEC would offer a simple and economical approach for the fabrication of efficient electrochemical biosensors.     

Determination of 5-aminolevulinic acid in blood plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 μl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26±0.08 μM (n=30) and 2.6±0.75 μM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain, and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Cloning and characterization of dichloromethane dehalogenase from Methylobacterium rhodesianum for dichloromethane degradation

The gene encoding dichloromethane dehalogenase from Methylobacterium rhodesianum was cloned. Bioinformatic analysis showed that dichloromethane dehalogenase gene sequence from M. rhodesianum is almost identical to the one from Methylobacterium extorquens, with only one base difference. Dichloromethane dehalogenase was subsequently expressed in Escherichia coli BL21 (DE3) and purified. It was found that enzyme activity in recombinant cells was 3 times higher than that in the wild-type M. rhodesianum. Further investigation showed that recombinant dichloromethane dehalogenase was most active at 40°C at pH 7–8, and its K_M was 10.96 mM when treated with dichloromethane as substrate. The fitted curve of dichloromethane degradation gave a V_max of 0.43 mM/h of in 0.01 M phosphate buffer. Degradation efficiency of dichloromethane reached 86.11% within 20 h. In addition, it was found that degradation efficiency of dichloromethane was highly associated with glutathione concentration, supporting the reports that glutathione functions as coenzyme of dichloromethane dehalogenase for dichloromethane degradation.     

Effects of fibre concentration of diets consisting of hay and slowly degradable concentrate on ruminal fermentation and digesta particle size in mid-lactation dairy cows

The objective of this study was to investigate the intestinal microbiota of growing kittens fed moderate- or high-protein diets using DNA-based qualitative and quantitative techniques. Kittens were weaned to a high-protein (HP; n = 7) or moderate-protein (MP; n = 10) diet at 8 weeks of age. Fresh faecal samples were collected at 8, 12, and 16 weeks of age. DNA was extracted and quantitative PCR used to quantify Bifidobacterium, Lactobacillus, Clostridium perfringens, and Escherichia coli concentrations. Denaturing gradient gel electrophoresis was performed to create a dendrogram and unrooted trees using Bionumerics 5.0 to identify similarity due to litter, age, or diet. Kittens fed HP had lower (p = 0.02) Bifidobacteria and Lactobacillus counts than MP-fed kittens. E. coli was lower (p = 0.02) in HP-fed kittens and tended to be affected by age (p = 0.09). Kittens were clustered by litter at 8 weeks of age, and then clustered by diet at 12 and 16 weeks of age. Our data suggest that faecal microbiota of growing kittens change after weaning and that dietary protein concentration affects E. coli, Bifidobacterium, and Lactobacillus populations. The relevance of these data in terms of intestinal health and disease remain to be determined and justifies further study.     

Phenol Biodegradation and Metal Removal by a Mixed Bacterial Consortium

This paper reports the tolerance and biodegradation of phenol by a heavy metal–adapted environmental bacterial consortium, known as consortium culture (CC). At the highest tolerable phenol concentration of 1200 mg/L, CC displayed specific growth rate of 0.04 h^?1, phenol degradation rate of 6.11 mg L^?1 h^?1 and biomass of 8.45 ± 0.35 (log₁₀ colony-forming units [CFU]/ml) at the end of incubation. Phenol was degraded via the ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific activity of 0.083 (µmol min^?1 mg^?1 protein). The different constituent bacterial isolates of CC preferentially grow on benzene, toluene, xylene, ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism involved in the degradation process. Microtox assay showed that phenol degradation was achieved without producing toxic dead-end metabolites. Moreover, lead (Pb) and cadmium (Cd) at the highest tested concentration of 1.0 and 0.1 mg/L, respectively, did not inhibit phenol degradation by CC. Simultaneous metal removal during phenol degradation was achieved using CC. These findings confirmed the dual function of CC to degrade phenol and to remove heavy metals from a mixed-pollutant medium.     

Highly sensitive amperometric immunosensor for the detection of Escherichia coli

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH₂-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl β-d-galactopyranoside (PAPG), the bacterial enzyme, β-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.     

EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-N-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Uridine 5′-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.     

Effect of increasing the dietary tryptophan to lysine ratio on plasma levels of tryptophan,kynurenine and urea and on production traits in weaner pigs experimentally infected with an enterotoxigenic strain of Escherichia coli‡

This experiment examined if immune system stimulation of weaner pigs, initiated by inoculation an enterotoxigenic strain of Escherichia coli (ETEC), increased the requirement for dietary tryptophan (Trp), modulated the inflammatory response, altered plasma levels of Trp and its metabolite kynurenine (Kyn) and effected post-weaning diarrhoea. Individually housed pigs (n = 72) weaned at 21 d of age were allocated to one of six treatments (n = 12) according to a two by three factorial arrangement of (1) with or without ETEC infection and (2) three dietary ratios of standardised ileal digestible (SID) Trp to lysine (Lys) (SID Trp:Lys) of 0.16, 0.20 or 0.24, in a completely randomised block design. Pigs had ad libitum access to diets (per kg 14.13 MJ ME, 12.4 g SID Lys, 195 g crude protein) for 3 weeks after weaning. Pigs were infected with ETEC (O149:K98:K88) at 72, 96 and 120 h after weaning and then bled on day 3, 11 and 19. An increased dietary Trp:Lys ratio increased plasma Trp and Kyn (p < 0.001) without effect of infection. On day 3, pigs fed 0.24 SID Trp:Lys had lower levels of plasma urea than at 0.20 Trp:Lys (p = 0.047) and on day 11, plasma urea was lower at 0.20 than at 0.16 SID Trp:Lys (p = 0.007). Infection increased (p = 0.039) the diarrhoea index and deteriorated faecal consistency from day 4–10 (p < 0.05). Treatments did not affect haptoglobin and acid soluble glycoprotein levels or daily gain and feed intake. However, 0.24 SID Trp:Lys improved (p = 0.021) feed efficiency without an effect of infection. In conclusion, in the absence of dietary antibiotic growth promotants, increasing the dietary SID Trp:Lys ratio to 0.24 improved feed conversion ratio after weaning and increased plasma levels of Trp and Kyn regardless of infection with E. coli.     

DNA Electrochemical Sensor for Detection of PRSS1 Point Mutation Based on Restriction Endonuclease Technique

To construct a restriction endonuclease based biosensor technology for PRSS1 genotyping. We designed a thiol-modified hairpin probe where the neck has EcoRI endonuclease recognition sites according to the PRSS1 gene c.410 C>T (p.T137 M) mutation and it was fixed on the gold electrode. Different charge generated by the binding of MB to phosphate groups of DNA before and after hybridization was used for distinguishing the different genotypes and quantity. This showed that the novel sensor can better distinguish the complementary sequence, single-base mismatches, and completely noncomplementary sequences, and the linear range for the logarithm was Y = –0.0242 X + 0.1574, R = 0.9912(Y = current, X = log target DNA concentration); the detection limit for DNA detection is estimated to be 50 fM.     

Batch and Fed-Batch Cultivation for Excretive Production of Human Epidermal Growth Factor (hEGF) with Recombinant E. Coli K12 System

Abstract

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32°C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Escherichia coli as a bioreporter in ecotoxicology

Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.     

Variation in chemical composition and physical characteristics of cereal grains from different genotypes

Genotypes of cereal grains, including winter barley (n = 21), maize (n = 27), oats (n = 14), winter rye (n = 22), winter triticale (n = 21) and winter wheat (n = 29), were assayed for their chemical composition and physical characteristics as part of the collaborative research project referred to as GrainUp. Genotypes of one grain species were grown on the same site, except maize. In general, concentrations of proximate nutrients were not largely different from feed tables. The coefficient of variation (CV) for the ether extract concentration of maize was high because the data pool comprised speciality maize bred for its high oil content. A subset of 8 barley, 20 rye, 20 triticale and 20 wheat samples was analysed to differ significantly in several carbohydrate fractions. Gross energy concentration of cereal grains could be predicted from proximate nutrient concentration with good accuracy. The mean lysine concentration of protein was the highest in oats (4.2 g/16 g N) and the lowest in wheat (2.7 g/16 g N). Significant differences were also detected in the concentrations of macro elements as well as iron, manganese, zinc and copper. Concentrations of arsenic, cadmium and lead were below the limit of detection. The concentration of lower inositol phosphates was low, but some inositol pentaphosphates were detected in all grains. In barley, relatively high inositol tetraphosphate concentration also was found. Intrinsic phytase activity was the highest in rye, followed by triticale, wheat, barley and maize, and it was not detectable in oats. Substantial differences were seen in the thousand seed weight, test weight, falling number and extract viscoelasticity characteristics. The study is a comprehensive overview of the composition of different cereal grain genotypes when grown on the same location. The relevance of the variation in composition for digestibility in different animal species will be subject of other communications.     

Bioluminescent bioreporter for assessment of arsenic contamination in water samples of India

In the present study the most efficient R-factor controlling the ars operon was selected after screening of 39 Escherichia coli isolates by minimum inhibitory concentration test (MIC) studies from water samples of different geographical locations of India. Among all, strain isolated from Hooghly River (West Bengal) was found to have maximum tolerance towards arsenic and was further used for the development of bioreporter bacteria. Cloning of the ars regulatory element along with operator-promotor and luxCDABE from Photobacteria into expression vector has been accomplished by following recombinant DNA protocols. The bioreporter sensor system developed in this study can measure the estimated range of 0.74–60 μg of As/L and is both specific and selective for sensing bioavailable As. The constructed bacterial biosensor was further used for the determination of arsenic ion concentration in different environmental samples of India.     

Application of artificial neural network model for the development of optimized complex medium for phenol degradation using <Emphasis Type="Italic">Pseudomonas pictorum</Emphasis> (NICM 2074)

Biodegradation of phenol using Pseudomonas pictorum (NICM 2074) a potential biodegradant of phenol was investigated for its degrading potential under different operating conditions. The neural network input parameter set consisted of the same set of four levels of maltose (0.025, 0.05, 0.075 g/l), phosphate (3, 12.5, 22 g/l), pH (7, 8, 9) and temperature (30°C, 32°C, 34°C) on phenol degradation was investigated and a Artificial Neural Network (ANN) model was developed to predict the extent of degradation. The learning, recall and generalization characteristic of neural networks was studied using phenol degradation system data. The efficiency of the model generated by the ANN, was tested and compared with the results obtained from an established second order polynomial multiple regression analysis (MRA). Further, the two models (ANN and MRA) were used to predict the percentage of degradation of phenol for blind test data. Performance of both the models were validated in the cases of training and test data, ANN was recommended based on the following higher coefficient of determination R ²; lower standard error of residuals and lower mean absolute percentage deviation.