Rapid and direct determination of glyphosate and aminomethylphosphonic acid in water using anion-exchange chromatography with coulometric detection

A simple, rapid, and low-cost coulometric method for direct detection of glyphosate and aminomethylphosphonic acid (AMPA) in water samples using anion-exchange chromatography and coulometric detection with copper electrode is presented. Under optimized conditions, the limits of detection (LODs) (S/N = 3) were 0.038 mu g ml(-1) for glyphosate and 0.24 mu g ml(-1) for AMPA, without any preconcentration method. The calibration curves were linear and presented an excellent correlation coefficient. The method was successfully applied to the determination of glyphosate and AMPA in water samples without any kind of extraction, clean-up, or preconcentration step. No interferent was found in the water, like this, the recovery was, practically, 100%. (c) 2008 Elsevier B.V. All rights reserved.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Single reaction interface flow system for chemiluminescent monitoring of mannitol based on its hydroxyl radical scavenger activity

A single reaction interface flow analysis (SIFA) system for the monitoring of mannitol in pharmaceutical formulations and human urine is presented. The developed approach takes advantage of the mannitol scavenger aptitude to inhibit the chemiluminescent reaction between luminol and myoglobin in the absence of H(2)O(2). The SIFA system facilitated the fully automation of the developed methodology, allowing the in-line reproducible handling of chemical species with a very short lifetime as is the case of the hydroxyl radical generated in the abovementioned luminol/myoglobin reaction. The proposed methodology allowed the determination of mannitol concentrations between 25 mmol L(-1) and 1 mol L(-1), with good precision (R.S.D. < 4.7%, n = 3) and a sampling frequency of about 60 h(-1). The procedure was applied to the determination of mannitol in pharmaceuticals and in human urine samples Without any pretreatment process. The results obtained for pharmaceutical formulations were statistically comparable to those provided by the reference method (R.D. < 4.6%); recoveries values obtained in the analysis of spiked urine samples (between 94.9 and 105.3% of the added amount) were also satisfactory. (C) 2008 Elsevier B.V. All rights reserved.

Biochemical properties of a beta-mannanase and a beta-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan, Its activity was optimal at pH 4.5. The K(m) value with galactoglucomannan as substrate was 0.50 mg ml (1). One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 degrees C and pH 8.0. Its K(m) value with birchwood xylan as substrate was 1.65 mg ml (1). Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn(+2), Fe(+3), and Cu(+2) were strong inhibitors for the mannanase. (C) 2008 Elsevier Ltd. All rights reserved.

Estimation of hexenuronic acids and kappa number in kraft pulps of Eucalyptus globulus by Fourier transform near infrared spectroscopy and multivariate analysis

Fourier transform near infrared (FT-NIR) spectroscopy was evaluated as an analytical too[ for monitoring residual Lignin, kappa number and hexenuronic acids (HexA) content in kraft pulps of Eucalyptus globulus. Sets of pulp samples were prepared under different cooking conditions to obtain a wide range of compound concentrations that were characterised by conventional wet chemistry analytical methods. The sample group was also analysed using FT-NIR spectroscopy in order to establish prediction models for the pulp characteristics. Several models were applied to correlate chemical composition in samples with the NIR spectral data by means of PCR or PLS algorithms. Calibration curves were built by using all the spectral data or selected regions. Best calibration models for the quantification of lignin, kappa and HexA were proposed presenting R-2 values of 0.99. Calibration models were used to predict pulp titers of 20 external samples in a validation set. The lignin concentration and kappa number in the range of 1.4-18% and 8-62, respectively, were predicted fairly accurately (standard error of prediction, SEP 1.1% for lignin and 2.9 for kappa). The HexA concentration (range of 5-71 mmol kg(-1) pulp) was more difficult to predict and the SEP was 7.0 mmol kg(-1) pulp in a model of HexA quantified by an ultraviolet (UV) technique and 6.1 mmol kg(-1) pulp in a model of HexA quantified by anion-exchange chromatography (AEC). Even in wet chemical procedures used for HexA determination, there is no good agreement between methods as demonstrated by the UV and AEC methods described in the present work. NIR spectroscopy did provide a rapid estimate of HexA content in kraft pulps prepared in routine cooking experiments.

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Grain-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest. (C) 2007 Elsevier Inc. All rights reserved.

In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698 alone and combined with galactooligosaccharides, milk and/or Bifidobacterium animalis subsp lactis Bb-12

Probiotic properties of Lactobacillus amylovorus DSM 16698 were previously demonstrated in piglets. Here, its potential as a human probiotic was studied in vitro, using the TIM-1 system, which is fully validated to simulate the human upper gastrointestinal tract. To evaluate the effect of the food matrix composition on the survival of L amylovorus DSM 16698 in TIM-1, the microorganism was inoculated alone or with prebiotic galactooligosaccharides (GOS), partially skimmed milk (PSM) and/or commercial probiotic Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12). Samples were collected from TIM-1 for six hours, at one-hour intervals and L amylovorus populations were enumerated on MRS agar plates with confirmation of identity of selected isolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. The cumulative survival for L amylovorus alone (control) was 30% at the end of the experiment (t = 6 h). Co-administration of L amylovorus with GOS. PSM and/or Bb-12 increased its survival in comparison with the control significantly from the 4th hour after ingestion onwards (P<0.05). Furthermore, by the use of High Performance Anion Exchange Chromatography, both L amylovorus and Bb-12 were observed to promptly degrade GOS compounds in samples collected from TIM-1, as assessed at t = 2 h. Hence, food matrix composition interfered with survival and growth of L. amylovorus during passage through TIM-1, providing leads towards optimization of probiotic properties in vivo. (C) 2011 Elsevier B.V. All rights reserved.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t(50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and characterization of a thermostable alpha-amylase produced by the fungus Paecilomyces variotii

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of alpha-amylase on Reagen (R) and Sigma (R) starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus

An extracellular alpha-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-d-glucopyranoside and presented an optimum temperature and pH of 65A degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60A degrees C, presenting t (50) of 90 min at 65A degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an alpha-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

In vivo degradation of banana starch: Structural characterization of the degradation process

This work reports the first ultrastructural investigation into the degradation process that starch granules isolated from bananas (cv. Nanicao) undergo during ripening. Starch granules from green bananas had a smooth surface, while granules from ripe bananas were more elongated with parallel striations, as revealed by CSLM and SEM. AFM images revealed that the first layer covering the granule surface is composed of a hard material and, as degradation proceeds, hard and soft regions seem to be repeated at regular intervals. WAXD patterns of banana starches were C-type, and the crystalline index was reduced during ripening. The B-/A-type ratio was increased, indicating the preferential degradation of the A-type allomorph. The branch-chain length distribution showed predominantly short chains of amylopectin (A and B1-chain). The fa/fb ratio was reduced during degradation, while amylose content was increased. The results allowed a detailed understanding of the changes that starch granules undergo during banana ripening. (C) 2010 Elsevier Ltd. All rights reserved.

Plantain and Banana Starches: Granule Structural Characteristics Explain the Differences in Their Starch Degradation Patterns

Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. alpha- and beta-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of alpha-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas.