Anion Chromatography Research Articles

Impact of biochemical properties on the gelation of EPS extracted from aerobic granules

Extracellular Polymeric Substances (EPS) extracted from aerobic granular sludge are a promising source of gel-forming biopolymers. Gaining insights on the charge distribution of these polymers was performed through a fractionation using anionic chromatography. A gelling factor (GF) based on the apparent gel volume and the reactivity of polymers to calcium was attributed to each eluted fraction. The GF values of the eluted anionic EPS correlated with their negative charge. A GF of 0.04 ± 0.03 is measured for molecules eluted at NaCl 0.25 M and a GF of 0.32 ± 0.02 for the molecules eluted at NaCl 0.75 M. However, upon reaching a very high negative charge (elution at NaCl 1 M), the gelation capacity decreases. The size distribution of gelling molecules was analyzed by Size Exclusion Chromatography. High Molecular Weight (MW) molecules are predominantly involved in gelation (between 65 % and 100 % of the high molecular weight fraction from each sample) while low MW molecules participation is relatively limited (lower than 40 % of the low molecular weight fraction from each sample). Moreover, infra-red spectroscopy analysis of the molecules before and after calcium addition showed that mostly proteins and polysaccharides contribute to the hydrogel formation. Colorimetric methods showed that around 60 % of the uronic acids present in the samples could form hydrogels whereas proteins’ participation varied between 0 % and 100 % depending on the fraction.Thus, modulating EPS extraction and purification methods to ensure the preservation of their molecular weight and the selection of appropriate negative charge could be a strategy for EPS valorization.

Unveiling New Arsenic Compounds in Plants via Tailored 2D-RP-HPLC Separation with ICP and ESI MS Detection.

Arsenic (As) speciation analysis is scientifically relevant due to the pivotal role the As chemical form plays in toxicity, which, in turn, directly influences the effect it has on the environment. The objective of this study was to develop and optimize a method tailored for studying As compounds in plant samples. Different extraction procedures and HPLC methods were explored to assess their efficiency, determine mass balance, and improve the resolution of compounds in the chromatograms. Conventionally applied anion-exchange chromatography facilitated the separation of well-documented As compounds in the extracts corresponding to 19 to 82% of As present in extracts. To gain insight into compounds which remain undetectable by anion chromatography (18 to 81% of As in the extracts), but still possibly metabolically relevant, we explored an alternative chromatographic approach. The procedure of sample purification and preconcentration through solid-phase extraction, facilitating the detection of those minor As compounds, was developed. The system was further refined to achieve an online 2D-RP-HPLC system, which was employed to analyze the extracts more comprehensively with ICP and ESI MS. Using this newly developed method, As(III)-phytochelatins, along with other arseno-thio-compounds, were detected and identified in extracts derived from the tree roots of seedlings grown in the presence of As(III) and As(V), and a group of arseno lipids was detected in the roots of plants exposed to As(V).

Chitinous material bioconversion by three new chitinases from the yeast Mestchnikowia pulcherrima

BackgroundChitinases are widely distributed enzymes that perform the biotransformation of chitin, one of the most abundant polysaccharides on the biosphere, into useful value-added chitooligosaccharides (COS) with a wide variety of biotechnological applications in food, health, and agricultural fields. One of the most important group of enzymes involved in the degradation of chitin comprises the glycoside hydrolase family 18 (GH18), which harbours endo- and exo-enzymes that act synergistically to depolymerize chitin. The secretion of a chitinase activity from the ubiquitous yeast Mestchnikowia pulcherrima and their involvement in the post-harvest biological control of fungal pathogens was previously reported.ResultsThree new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris to about 91, 90 and 71 mU ml− 1, respectively. The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, increased 2-times their activities using 1 mM of Mn2+ and hydrolysed different types of commercial chitosan. The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product.ConclusionsThree new chitinases from the yeast M. pulcherrima were molecularly characterized and their enzymatic and structural characteristics analysed. These enzymes transformed chitinous materials to fully and partially acetylated COS through different modes of splitting, which make them interesting biocatalysts for deeper structural-function studies on the challenging enzymatic conversion of chitin.

Oxidative degradation of Piperazine (PZ) in aqueous KOH/K2CO3 solutions

KOH and K2CO3 aqueous solutions have demonstrated potential for CO2 absorption processes due to their low toxicity, non-volatility of the capture agent and resistance to degradation. However, their absorption kinetics are typically slower than those of primary and secondary amines, and therefore promotors may be needed to allow competitive absorption rates and thus absorber heights. Here, we selected piperazine (PZ) as a promoter and investigated its oxidative degradation when blended with KOH or K2CO3. The changes in PZ concentration were analyzed by gas chromatography–mass spectrometry (GC–MS), while the degradation products were analyzed by GC–MS and anion chromatography. The results showed that PZ oxidative degradation followed the order KOH/PZ > PZ > K2CO3/PZ indicating that KOH enhanced PZ oxidation while K2CO3 inhibited PZ oxidation. Therefore, KOH/PZ may still be a promising solvent with appropriate CO2 loading as the CO2 protects the PZ from hydroxyl and oxygen radical attacks. The degradation products are similar across the blends and identified as Ethylenediamine (EDA), 2-oxopiperazine (OPZ), Formylpiperazine (FPZ), formate, acetate and oxalate, with formate being the most abundant product for all blends. Furthermore, the PZ first-order rate constants for all solutions were provided, and the oxidation degradation pathway of PZ-based solutions was proposed.

Separation of bispecific antibody related impurities with mixed-mode chromatography

The complex structure of bispecific antibody molecules and the rapid increase in upstream expression pose a significant challenge for downstream purification. Compared to mAbs, bsAbs typically contain a higher proportion of aggregates and fragments that are difficult to remove. Extensive study and optimization are often required because the properties of impurities are close to the target molecules. In this investigation, ion exchange resins and mixed-mode resins were used to evaluate the effectiveness in removing byproducts. Mixed-mode anion chromatography was found to be effective in polishing step of bsAbs with strong hydrophobicity. Hydroxyapatite chromatography using arginine hydrochloride as an eluent provided excellent separation performance compared to that of cation exchange resins and conventional mixed-mode resins. The separation process was optimized and verified in a 200 L pilot batch. The final products were obtained with excellent purity (99.0 %) and yield (80.70 %). This work provides a new idea for high-yield polishing in downstream processing, which improves the economics of large-scale production.

MP10-15 ALKALINE WATER: HELP OR HYPE FOR URIC ACID STONE PATIENTS?

You have accessJournal of UrologyCME1 Apr 2023MP10-15 ALKALINE WATER: HELP OR HYPE FOR URIC ACID STONE PATIENTS? Paul Piedras, Amanda McCormac, Seyed Amiryaghoub Lavasani, Minh-Chau Vu, Rohit Bhatt, Kalon Morgan, Andrei Cumpanas, Zachary Tano, John Asplin, Sohrab N. Ali, Pengbo Jiang, Jaime Landman, Ralph V. Clayman, and Roshan M. Patel Paul PiedrasPaul Piedras More articles by this author , Amanda McCormacAmanda McCormac More articles by this author , Seyed Amiryaghoub LavasaniSeyed Amiryaghoub Lavasani More articles by this author , Minh-Chau VuMinh-Chau Vu More articles by this author , Rohit BhattRohit Bhatt More articles by this author , Kalon MorganKalon Morgan More articles by this author , Andrei CumpanasAndrei Cumpanas More articles by this author , Zachary TanoZachary Tano More articles by this author , John AsplinJohn Asplin More articles by this author , Sohrab N. AliSohrab N. Ali More articles by this author , Pengbo JiangPengbo Jiang More articles by this author , Jaime LandmanJaime Landman More articles by this author , Ralph V. ClaymanRalph V. Clayman More articles by this author , and Roshan M. PatelRoshan M. Patel More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003225.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The consumption of alkaline water, an electrolyzed mineral water with an average pH of 8-10, has been steadily increasing globally as proponents claim it to be a healthier alternative to tap water. Urinary alkalinization therapy is frequently prescribed in patients with uric acid nephrolithiasis but given low compliance there has been interest in alkaline water as a potential therapeutic alternative. For any fluid to have an alkalinizing effect on urine, it must contain organic anions such as citrate and bicarbonate. We analyzed five commercially available alkaline water products to assess their electrolyte and organic anion composition. METHODS: Five commercially available alkaline water brands (Essentia®, Smart Water® Alkaline, Great Value™ Hydrate Alkaline Water, Body Armor® SportWater, and Perfect Hydration®) were subjected to anion chromatography, acid titration, and direct chemical measurements to determine the mineral contents of each product. The alkali content of each brand of alkaline water was then contrasted with the alkali content of potassium citrate, sodium bicarbonate and other over the counter organic and synthetic beverages. RESULTS: The pH levels of the five alkaline waters ranged from 9.69-10.15; however, the electrolyte content was minimal, and the physiologic alkali content was <1 mEq (Table 1). The alkali content of all five brands of alkaline water was considerably lower than potassium citrate, sodium bicarbonate, and other common over the counter organic and synthetic beverages (Table 2). CONCLUSIONS: For uric acid stone patients, commercially available alkaline water has negligible alkali content and thus would likely provide no added benefit over consumption of an equal volume of tap water. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e120 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Piedras More articles by this author Amanda McCormac More articles by this author Seyed Amiryaghoub Lavasani More articles by this author Minh-Chau Vu More articles by this author Rohit Bhatt More articles by this author Kalon Morgan More articles by this author Andrei Cumpanas More articles by this author Zachary Tano More articles by this author John Asplin More articles by this author Sohrab N. Ali More articles by this author Pengbo Jiang More articles by this author Jaime Landman More articles by this author Ralph V. Clayman More articles by this author Roshan M. Patel More articles by this author Expand All Advertisement PDF downloadLoading ...

Production and characterization of exopolysaccharides from salinity-induced Auxenochlorella protothecoides and the analysis of anti-inflammatory activity

The set scenario of this work was to investigate the production, physicochemical characteristics, and anti-inflammatory activities of exopolysaccharides from salinity-induced Auxenochlorella protothecoides. The results demonstrated that 10 ‰ salinity manipulation endowed preferable exopolysaccharide production by A. protothecoides. Under this salinity stress, ACPEPS1A and ACPEPS2A were purified from exopolysaccharide production by anion chromatography and molecular exclusion chromatography. ACPEPS1A exhibited a molecular weight (Mw) of 132 kDa and mainly consisted of galactose. ACPEPS2A was a heteropolysaccharide with an Mw of 170 kDa and the main monosaccharides of galactose and rhamnose with separate molar percents of 42.41 % and 35.29 %, respectively. FTIR, 1H and 13C NMR supported that monosaccharide components of ACPEPS1A and ACPEPS2A possessed both α- and β-configuration pyranose rings. Further evidence indicated that ACPEPS1A and ACPEPS2A could effectively inhibit the inflammatory response in lipopolysaccharide (LPS) induced RAW264.7 cells by quenching inflammatory factor levels such as ROS, iNOS, TNF-α, and IL-6. The potential anti-inflammatory possibilities were that the monosaccharides of ACPEPS1A and ACPEPS2A possessed higher affinity with receptors on the macrophage surface than LPS and hampered LPS-induced inflammation. The findings of this work would favor innovative applications of exopolysaccharides from microalgae in complementary medicines or functional foods.

Standardized method for mechanistic modeling of multimodal anion exchange chromatography in flow through operation

Multimodal chromatography offers an increased selectivity compared to unimodal chromatographic methods and is often employed for challenging separation tasks in industrial downstream processing (DSP). Unfortunately, the implementation of multimodal polishing into a generic downstream platform can be hampered by non-robust platform conditions leading to a time and cost intensive process development. Mechanistic modeling can assist experimental process development but readily applicable and easy to calibrate multimodal chromatography models are lacking. In this work, we present a mechanistic modeling aided approach that paves the way for an accelerated development of anionic mixed-mode chromatography (MMC) for biopharmaceutical purification. A modified multimodal isotherm model was calibrated using only three chromatographic experiments and was employed in the retention prediction of four antibody formats including a Fab, a bispecific, as well as an IgG1 and IgG4 antibody subtype at pH 5.0 and 6.0. The chromatographic experiments were conducted using the anionic mixed-mode resin Capto adhere at industrial relevant process conditions to enable flow through purification. An existing multimodal isotherm model was reduced to hydrophobic interactions in the linear range of the adsorption isotherm and successfully employed in the simulation of six chromatographic experiments per molecule in concert with the transport dispersive model (TDM). The model reduction to only three parameters did prevent structural parameter non-identifiability and enabled an analytical isotherm parameter determination that was further refined by incorporation of size exclusion effects of the selected multimodal resin. During the model calibration, three linear salt gradient elution experiments were performed for each molecule followed by an isotherm parameter uncertainty assessment. Lastly, each model was validated with a set of step and isocratic elution experiments. This standardized modeling approach facilitates the implementation of multimodal chromatography as a key unit operation for the biopharmaceutical downstream platform, while increasing the mechanistic insight to the multimodal adsorption behavior of complex biologics.

Purification of RgpA from external outer membrane vesicles of Porphyromonas gingivalis

IntroductionPurification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV). MethodsNative RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719–865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA. ResultsDouble anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (Log10) 7.4 ± 0.08/mL was of 12.6% (2 mg/mL), with 3.2 FU/μg of amidolytic activity. ConclusionsThis protocol allows purification of native RgpA from OMV that retains protease activity.

Polyelectrolyte-grafted anion exchangers with hydrophilic intermediate layers for ion chromatography

The hydrophobicity of polymer-based anion exchangers is a persistent problem in suppressed anionic chromatography (SAC) due to its adverse effect on chromatographic performance. Herein we describe polyelectrolyte-grafted anion exchangers with modified hydrophilic intermediate layers. The anion exchangers were functionalized by successively grafting a linear cationic condensation polymer (LCCP), a preprepared polyelectrolyte. The carboxylic/hydroxylic intermediate layers formed during thiol-radical-mediated polymerization exert distinct effects on the phase capacity and hydrophobicity. The separation of typical inorganic anions, polarizable anions, and organic acids shows that the anion exchangers display good performance in SAC mode.

Preparation of Nereid oligopeptide and investigation of the mechanism underlying the induction of apoptosis in human lung cancer H1299 cells.

In the present study, oligopeptides from Nereid (Perinereis aibuhitensis) were prepared via enzymatic hydrolysis, and the mechanism underlying the induction of apoptosis in H1299 cells was investigated. According to the analysis of the inhibition rate on proliferation, alkaline protease demonstrated the best enzymatic efficiency. The optimal conditions for hydrolysis were as follows: 50°C and pH 10 for 6 h; a material-to-liquid ratio of 1:1 (g/ml); and addition of 400 U/g enzyme. The hydrolysates were purified using ultrafiltration, anion chromatography, gel filtration chromatography, and high-performance liquid chromatography. The Nereid oligopeptide (NOP), with a molecular weight of 841 kDa and an amino acid sequence of glutamine-isoleucine-asparagine-glutamine-histidine-leucine, was obtained. NOP inhibited the proliferation of H1299 cells in a time- and dose-dependent manner. Morphological changes and apoptosis were also induced by NOP in H1299 cells. The western blot analysis revealed that the B-cell lymphoma 2/Bcl-2 associated X (Bcl-2/Bax) ratio was reduced by 24.7% in the NOP treatment group compared with the control group. The relative expression levels of cleaved caspase-9 (cleaved-CASP9) and cleaved caspase-3 (cleaved-CASP3) in the NOP treatment group were 2.55- and 1.71-fold higher than those measured in the control group, respectively. These results suggested that NOP exerts antitumor effects by influencing the proliferation and apoptosis of H1299 cells.

Retention of inorganic anions using mesoporous zirconia spheres modified with anion-exchange groups as the stationary phase for ion chromatography.

A zirconia (ZrO2) stationary phase with a chemically fixed silane coupling agent (trimethyl [3-(trimethoxysilyl)propyl]ammonium chloride; TMA), which possesses quaternary ammonium functional groups, is evaluated as a separation column for ion chromatography (IC) of anions. The selectivity for anions varies depending on the amount of TMA immobilized on the ZrO2. The TMA-ZrO2, with an anion-exchange capacity of 17 ± 3µeq/g, shows an anion-exchange reaction that involves the specific retention of fluoride ion on ZrO2. The TMA-ZrO2 exhibits a decrease of the anion resolution with an increase of the eluent pH and an enhancement of the selective separation of fluorideion with an increase of the column temperature. Through this study, the TMA-ZrO2 stationary phase shows potential as a new medium for ion separation.

Bioleaching Coal Gangue with a Mixed Culture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

A mixed culture of A. ferrooxidans and A. thiooxidans isolated from a coal gangue dump was used to bioleach coal gangue in a column reactor to investigate the leaching of elements. The changes of metal ions (Fe, Mn and Cr) and sulfate in the leaching solution, elemental composition, mineral components and sulfur speciation of the coal gangue before and after bioleaching were analyzed by atomic absorption, anion chromatography, XRF, XRD and XPS. The results show that the mixed culture could promote the release of metal ions in coal gangue, with a leaching concentration of Fe &gt; Mn &gt; Cr. EC and Eh have significantly increased with the increase of metal ion concentrations in the leaching solution. XRF analyses show that the contents of Fe, Mn and S decreased in coal gangue after bioleaching. XRD results suggest that the bioleaching has impacts on minerals in coal gangue, particularly the Fe-containing components. XPS analyses show that sulfur speciation in the raw gangue samples was associated with sulfate, dibenzothiophene and pyrite sulfur. After continuous leaching by the mixed culture, the total sulfur, pyrite sulfur and sulfate sulfur in coal gangue decreased from 2.06% to 1.18%, 0.66% to 0.14% and 1.02% to 0.52%. The desulfurization rates of the pyrite and sulfate were 78.79% and 49.02 %. It is concluded that the mixed culture of these two microorganisms could effectively leach metals from coal gangue coupling with the oxidation of sulfide to sulfate. This study has provided fundamental information as a potential application in the recovery of valuable metals from coal gangue or environmental remediation related to gangue in the future.

A systematic high-throughput phenotyping assay for sugarcane stalk quality characterization by near-infrared spectroscopy

BackgroundSugarcane (Saccharum officinarum L.) is an economically important crop with stalks as the harvest organs. Improvement in stalk quality is deemed a promising strategy for enhancing sugarcane production. However, the lack of efficient approaches for systematic evaluation of sugarcane germplasm largely limits improvements in stalk quality. This study is designed to develop a systematic near-infrared spectroscopy (NIRS) assay for high-throughput phenotyping of sugarcane stalk quality, thereby providing a feasible solution for precise evaluation of sugarcane germplasm.ResultsA total of 628 sugarcane accessions harvested at different growth stages before and after maturity were employed to take a high-throughput assay to determine sugarcane stalk quality. Based on high-performance anion chromatography (HPAEC-PAD), large variations in sugarcane stalk quality were detected in terms of biomass composition and the corresponding fundamental ratios. Online and offline NIRS modeling strategies were applied for multiple purpose calibration with partial least square (PLS) regression analysis. Consequently, 25 equations were generated with excellent determination coefficients (R2) and ratio performance deviation (RPD) values. Notably, for some observations, RPD values as high as 6.3 were observed, which indicated their exceptional performance and predictive capability.ConclusionsThis study provides a feasible method for consistent and high-throughput assessment of stalk quality in terms of moisture, soluble sugar, insoluble residue and the corresponding fundamental ratios. The proposed method permits large-scale screening of optimal sugarcane germplasm for sugarcane stalk quality breeding and beyond.

Investigations on historical monuments\u2019 deterioration through chemical and isotopic analyses: an Italian case study

In this paper, we analysed the efflorescences present in the frescos of a monumental complex named S. Pietro a Corte situated in the historic centre of Salerno (Campania, Italy). The groundwater of the historic centre is fed by two important streams (the Rafastia and the Fusandola) that can be the sources of water penetration. The aims of this work are to (i) identify the stream that reaches the ancient frigidarium of S. Pietro a Corte and (ii) characterize the efflorescences on damaged frescos in terms of chemical nature and sources. In order to accomplish the first aim, the water of the Rafastia river (7 samples) and the water of the Fusandola river (7 samples) were analysed and compared with the water of a well of the Church (7 samples). The ionic chromatography measurements on the water samples allowed us to identify the Rafastia as the river that feeds the ancient frigidarium of S. Pietro a Corte. To investigate the nature and the origin of the efflorescences (our second aim), anionic chromatography analyses, X-ray diffraction measurements, and the isotopic determination of nitrogen were performed on the efflorescences (9 samples) and the salts recovered from the well (6 samples). Results of these analyses show that efflorescences are mainly made of potassium nitrate with a δ15N value of + 9.3 ± 0.2‰. Consequently, a plausible explanation for their formation could be the permeation of sewage water on the walls of the monumental complex.

Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

The present study describes the cloning of the cellobiohydrolase gene from a thermophilic bacterium Clostridium clariflavum and its expression in Escherichia coli BL21(DE3) utilizing the expression vector pET-21a(+). The optimization of various parameters (pH, temperature, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, time of induction) was carried out to obtain the maximum enzyme activity (2.78 ± 0.145 U ml−1) of recombinant enzyme. The maximum expression of recombinant cellobiohydrolase was obtained at pH 6.0 and 70 °C respectively. Enzyme purification was performed by heat treatment and immobilized metal anionic chromatography. The specific activity of the purified enzyme was 57.4 U mg−1 with 35.17% recovery and 3.90 purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cellobiohydrolase was 78 kDa. Among metal ions, Ca2+ showed a positive impact on the cellobiohydrolase enzyme with increased activity by 115%. Recombinant purified cellobiohydrolase enzyme remained stable and exhibited 77% and 63% residual activity in comparison to control in the presence of n-butanol and after incubation at 80 °C for 1 h, respectively. Our results indicate that our purified recombinant cellobiohydrolase can be used in the biofuel industry.

Isolation, characterization and regulation of moonlighting proteases from Candida glabrata cell wall

Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10–50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.

Biochemical and functional characterization of a new recombinant phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.

Validation of a radiochemical method for the determination of 55Fe and 63Ni in water and steel samples from decommissioning activities

We present the validation of a radiochemical procedure for the determination of 55Fe and 63Ni in water and steel samples from nuclear decommissioning activities. The in-house validation was an essential requirement of the accreditation to ISO/IEC 17025. A combination of co-precipitation, anion chromatography and extraction chromatography steps was used for separating and purifying both radionuclides. The activities were measured by liquid scintillation counting. Due to the lack of reference materials, the method was validated by analysing simulated samples and by the standard addition technique. The parameters precision/accuracy, repeatability/linearity, selectivity/specificity, decision threshold, detection limit and uncertainty of the method were evaluated.

Selection of a versatile Lactobacillus plantarum for wine production and identification and preliminary characterisation of a novel histamine‐degrading enzyme

SummaryIn this study, a desirable and versatile Lactobacillus plantarum strain possessing great ability of L‐malic acid consumption, biogenic amine degradation and resistance to wine harsh environment was obtained through successive screenings. Malolactic fermentation (MLF) in contaminated grape wine and cherry wine (supplemented with histamine, tyramine and cadaverine) conducted by this strain was finished within 24 and 18 days, respectively, and the concentration of histamine, tyramine and cadaverine was decreased by over 57% following MLF. The enzyme from L. plantarum responsible for the amine degradation was purified to homogeneity by four steps including cell disruption, ammonium sulphate fractionation, an anion chromatography and a gel filtration chromatography. Such enzyme was identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) after N‐terminal sequence analysis and protein Blast, a tetramer enzyme with a subunit molecular weight of 36 kDa. The optimum values of pH and temperature of this enzyme were at pH 7.5 and 40 °C, and stable between pH 5.5–8.5 and 30–50 °C.