Partial depolymerization of pectin by a photochemical reaction

Complex heterogeneous polysaccharides that comprise pectin were partially depolymerized by a photochemical reaction using ultraviolet light in the presence of titanium dioxide catalyst. In a period of 6 h at pH 7, this UV/TiO₂ process decreased the average molecular weight of pectin from 400 kDa to 200 kDa. The characterization of the partially depolymerized pectin, which was fractionated by size-exclusion chromatography, was performed by ¹H NMR spectroscopy, and the spectra obtained showed that the resulting oligosaccharides and polysaccharides maintained the intact core structure of pectin. The monosaccharide content and depolymerization profile were determined by high-performance anion-exchange chromatography coupled with pulsed amperometric detection. This controlled photochemical depolymerization technique might be useful for preparation of pectin oligosaccharides as an ingredient in food and pharmaceutical products.     

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and ¹³C NMR. Optimum conditions afford samples at 50–80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for ¹³C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for M_W > 4 kDa. The M_W values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples. Received: 10 July 1997 / Accepted: 12 November 1997     

Immunodetection of a 90 000-Mr polypeptide related to yeast plasma membrane ATPase in plasma membrane from maize shoots

Maize shoot plasma membranes were prepared using either polyethyleneglycol (PEG)-dextran phase partition or centrifugation through a 30% sucrose cushion. The ATPase specific activity of membranes obtained with the phase partition method (1.4 μmol P_i · min⁻¹ · mg⁻¹ protein) was twice that of those prepared with the sucrose cushion method. After solubilization by lysolecithin and precipitation by ammonium sulfate, ATPase activities of the order of 3.0–3.5 μmol P_i · min⁻¹ · mg⁻¹ were obtained. A polypeptide of M_r = 90 000 was enriched during ATPase purification.Antibodies against pure plasma membrane ATPase from Saccharomyces cerevisiae inhibited the plant ATPase activity. Immunodetection during purification of the plant enzyme strongly supported the conclusion that the polypeptide of M_r = 90 000 belongs to plant plasma membrane ATPase.     

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr⁻¹. The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr⁻¹, strain QU 50 produced 42.6 g L⁻¹ of l -LA with a yield of 0.912 g g⁻¹-consumed sugars, LA yield of 0.655 g g⁻¹ based on mixed sugar-loaded, and a productivity of 4.31 g L⁻¹ hr⁻¹ from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L⁻¹ with LA yield of 0.944 g g⁻¹-consumed sugar and LA productivity of 6.49 ± 0.357 g L⁻¹ hr⁻¹. Besides this, a dramatic increase in LA yield of 0.927 g g⁻¹ based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.     

In vitro and in vivo pharmacological characterization of the novel neuropeptide S receptor ligands QA1 and PI1

The pharmacological activity of the novel neuropeptide S (NPS) receptor (NPSR) ligands QA1 and PI1 was investigated. In vitro QA1 and PI1 were tested in calcium mobilization studies performed in HEK293 cells expressing the recombinant mouse (HEK293_mNPSR) and human (HEK293_hNPSRIle107 and HEK293_hNPSRAsn107) NPSR receptors. In vivo the compounds were studied in mouse righting reflex (RR) and locomotor activity (LA) tests. NPS caused a concentration dependent mobilization of intracellular calcium in the three cell lines with high potency (pEC₅₀ 8.73–9.14). In inhibition response curve and Schild protocol experiments the effects of NPS were antagonized by QA1 and PI1. QA1 displayed high potency (pK_B 9.60–9.82) behaving as a insurmountable antagonist. However in coinjection experiments QA1 produced a rightward swift of the concentration response curve to NPS without modifying its maximal effects; this suggests that QA1 is actually a slow dissociating competitive antagonist. PI1 displayed a competitive type of antagonism and lower values of potencies (pA₂ 7.74–8.45). In vivo in mice NPS (0.1 nmol, i.c.v.) elicited arousal promoting action in the RR assay and stimulant effects in the LA test. QA1 (30 mg kg⁻¹) was able to partially counteract the arousal promoting NPS effects, while PI1 was inactive in the RR test. In the LA test QA1 and PI1 only poorly blocked the NPS stimulant action. The present data demonstrated that QA1 and PI1 act as potent NPSR antagonists in vitro, however their usefulness for in vivo investigations in mice seems limited probably by pharmacokinetic reasons.     

Purification of nitrate reductase from spinach (Spinacea oleracea L.) by immunoaffinity chromatography using a monoclonal antibody

NADH-Nitrate reductase (EC 1.6.6.1) from spinach (Spinacea oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat γ globulin prevented non-specific binding. The enzyme, released with 1 M KNO₃, was purified 1550-fold to a specific activity of 24.8 μmol NO₂⁻ produced min⁻¹, mg protein⁻¹ with a recovery of 60% of applied NADH-NR activity. Proteolytically ‘nicked’ subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5′-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87).     

Utilization of bicarbonate by apple fruit Phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC), was partially purified from apple fruit cv Golden Delicious. Kinetic values for PEP and HCO⁻₃ suggest a capacity for efficient carbon dioxide refixation. PEPC activity was maximal between 5–10 mM carbonate (HCO⁻₃) and inhibition was observed above 10 mM HCO⁻₃. In conditions of PEP-saturation, HCO⁻₃ inhibition of apple fruit PEPC activity appeared non-competitive with respect to PEP and was partially reversible.     

Use of liquid chromatography to measure chlorite production by Nitrobacter winogradskyi NRCC26538

A high-pressure liquid chromatography (HPLC) technique, previously developed for nitrite (NO⁻₂) and nitrate (NO⁻₃) measurements [3], was used to measure chlorite (ClO⁻₂) production by Nitrobacter winogradskyi. The determination of ClO⁻₂ by HPLC involves monitoring the column effluent with a UV detector at 214 or 254 nm. Although the absorbance of ClO⁻₂ at 214 nm was about 5 times greater than at 254 nm, interference from other compounds in the culture filtrates of N. winogradskyi contributed to an unstable detector signal. The detection limit at 254 nm for ClO⁻₂ in deionized water was about 1 μM.The measurement of ClO⁻₂ in N. winogradskyi culture filtrates was done with detection at 254 nm. The maximum concentration of ClO⁻₂ produced by anaerobically incubated cell suspensions of N. winogradskyi was about 80 μM.     

Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH)₂D] in human serum has been developed. Whereas small amounts of [³H]24,25-(OH)₂D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D₃ ([³H]25-OHD₃) can be used as the assay tracer. Since 25-OHD₃ and 24,25-(OH)₂D₃ are equipotent in their competitive displacement of [³H]25-OHD₃ from rat serum, 25-OHD₃ can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH)₂D by batch elution. The purity of biosynthesized [³H]24,25-(OH)₂D₃ and the 24,25-(OH)₂D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH)₂D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH)₂D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.     

Elimination of hydrophobic volatile organic compounds in fungal biofilters: reducing start-up time using different carbon sources

Fungal biofilters have been recently studied as an alternative to the bacterial systems for the elimination of hydrophobic volatile organic compounds (VOC). Fungi foster reduced transport limitation of hydrophobic VOCs due to their hydrophobic surface and extended gas exchange area associated to the hyphal growth. Nevertheless, one of their principal drawbacks is their slow growth, which is critical in the start‐up of fungal biofilters. This work compares the use of different carbon sources (glycerol, 1‐hexanol, wheat bran, and n‐hexane) to reduce the start‐up period and sustain high n‐hexane elimination capacities (EC) in biofilters inoculated with Fusarium solani. Four parallel experiments were performed with the different media and the EC, the n‐hexane partition coefficient, the biomass production and the specific consumption rate were evaluated. Biofilters were operated with a residence time of 1.3 min and an inlet n‐hexane load of 325 g m⁻³_reactor h⁻¹. The time to attain maximum EC once gaseous n‐hexane was fed was reduced in the three experiments with alternate substrates, as compared to the 36 days needed with the control where only n‐hexane was added. The shortest adaptation period was 7 days when wheat bran was initially used obtaining a maximum EC of 160 g m⁻³_reactor h⁻¹ and a critical load of 55 g m⁻³_reactor h⁻¹. The results were also consistent with the pressure drop, the amount of biomass produced and its affinity for the gaseous n‐hexane, as represented by its partition coefficient. Biotechnol. Bioeng. 2011; 108:758–765. © 2010 Wiley Periodicals, Inc.     

Protective effect of a freshwater alga, <Emphasis Type="Italic">Spirogyra</Emphasis> sp., against lipid peroxidation in vivo zebrafish and purification of antioxidative compounds using preparative centrifugal partition chromatography

A freshwater alga, Spirogyra sp., collected in shallow ponds in South Korea, was evaluated for its antilipid peroxidative effect against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced in vivo zebrafish, and antioxidative compounds from the alga were efficiently identified using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS⁺) online high-performance liquid chromatography (HPLC) and preparative centrifugal partition chromatography. The ethyl acetate fraction of Spirogyra sp. (SPE) in each fraction showed the strongest 2,2-diphenyl-1-picrylhydrazyl scavenging activity and significantly scavenged 2′,7′-dichlorodihydrofluorescein diacetate and diphenyl-1-pyrenylphosphine fluorescence, respectively, in AAPH-induced zebrafish embryo without any cytotoxicity in the concentrations between 25 and 50 μg mL^?1. The two main antioxidative compounds in SPE were confirmed by ABTS⁺ online HPLC and were identified as gallic acid and methyl gallate, respectively, by HPLC–diode array detection (DAD)–electrospray ionization (ESI)/mass spectrometry (MS), ¹H- and ¹³C-NMR. We conclude therefore that Spirogyra sp. is rich in gallic acid and methyl gallate, and it might be useful as a strong antilipid peroxidation material.     

Influence of lignin on the methanization of lignocellulosic wastes

The effect on anaerobic digestion of reducing the lignin content of vine shoots to 1% (w/w), by treatment with sodium chlorite in an acid medium at 80°C, is reported. The yields of methane obtained were 240 ml of CH₄ g⁻¹ of VS (volatile solids) fed for untreated vine shoots, and 370 ml of CH₄ g⁻¹ of VS fed for treated vine shoots. A mathematical model was used to calculate the kinetic parameters H and μ, and the increased biodegradability of the substrate in which lignin had been removed was confirmed. A study of the mass balances of the process under optimum conditions (temperature = 35°C; loading rate of 1 g litre⁻¹ digester day⁻¹) enabled the percentage of degraded cellulose to be calculated (35·5% for untreated vine shoots, 81·5% for the treated vine shoots), as were the volumes of biogas and methane produced per gram of VS introduced (VS₁) and degraded. The blocking effect of lignin on the methanization process was confirmed.     

Exacerbated intestinal inflammation in P2Y6 deficient mice is associated with Th17 activation

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y₆ receptor. P2Y₆ activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y₆ expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y₆ would participate to the establishment of inflammation in this disease. To address this, we used P2Y₆ deficient (P2ry6^‐−/−) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y₆ deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6^−/− mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6^−/− mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1β in P2ry6^−/− colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6^−/− mice from DSS-induced colitis. Taken together, in the absence of P2Y₆, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y₆ expressed on leukocytes in intestinal inflammation.     

Partitioning of oxygen consumption between “maintenance” and “growth” in developing herring Clupea harengus (L.) embryos

Growth and oxygen consumption was measured in developing herring Clupea harengus (L.) embryos. By considering the variations in oxygen consumption with embryonic size and growth rate, an attempt was made to partition oxygen consumption between growth related and growth unrelated (i.e., “maintenance”) processes. The metabolic cost of growth was estimated as ≈ 150 ng O₂ · μg dry wt tissue formed⁻¹. This estimate compares favourably with the biochemical estimate of the costs of transport and net biosynthesis. The “maintenance” component was proportional to embryonic mass (77 ng O₂ · μg⁻¹· d⁻¹). Over the entire embryonic period, growth processes were responsible for ≈ 25% of the cumulated oxygen consumption.     

A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography

Introduction – Eisenia bicyclis (Kjellman) Setchell (Laminariaceae) is a common brown alga that inhabits around the coast of Korea, Japan and China. It contains fucoxanthin, a major carotenoid of brown algae which shows a variety of pharmaceutical functions. Objective – The aim of this investigation was the quantification and preparative isolation of fucoxanthin from fresh E. bicyclis using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – The fucoxanthin fraction (Fuco fraction) was prepared by solvent partition method from the acetone extract of fresh E. bicyclis. Fuco fraction was used for CPC using a two‐phase solvent system of n‐hexane–ethyl acetate–ethanol–water (5:5:7:3, v/v/v/v). The flow rate of mobile phase was 2 mL/min with descending mode while rotating at 1000 rpm. The eluate was monitored at 410 nm. The content and structure of fucoxanthin in the CPC fraction were confirmed with HPLC, UV, APCI/MS and NMR spectra. Results – A preparative CPC yielded 20 mg of fucoxanthin (87% recovery from Fuco fraction) in a two‐step separation from 516 mg of Fuco fraction containing 4.59% fucoxanthin. The purity of the isolated fucoxanthin was about 81% in the first CPC step and over 98% in the second CPC step based on the calibration curve. The isolated fucoxanthin was identified as all‐trans‐fucoxanthin with APCI/MS (parent ion at m/z 641 [M + H ? H₂O]⁺) and ¹H, ¹³C and 2‐D NMR spectra. Conclusion – High‐purity fucoxanthin was successfully isolated from fresh E. bicyclis, suggesting further potential applications in the industrial use of this valuable carotenoid. Copyright © 2011 John Wiley & Sons, Ltd.     

Studies on the localization and properties of rat duodenal HCO3−-ATPase with special relation to alkaline phosphatase

Brush-border membrane fractions were isolated from rat duodenum. Purity and integrity of the fraction was confirmed by electron microscopy, enzymic analysis and demonstration of Na⁺-dependent glucose uptake. The membranes were enriched 15-fold in alkaline phosphatase and α-glucosidase and 6-fold in HCO₃⁻-ATPase activities. Assays of latent activity indicated that these enzymes were predominantly localised to the external aspect of the microvillus membrane. The enzymes were solubilised and subjected to analysis by gel filtration, ion exchange and phenylboronate chromatography. No separation of alkaline phosphatase and HCO₃⁻-ATPase was obtained and it is suggested that they reflect the same enzyme activity. The apparent activation by HCO₃⁻ was investigated, and was found to be due to shifts in the pH dependency of the activity due to changes in ionic strength.     

Belactins A and B,New Serine Carboxypeptidase Inhibitors Produced by Actinomycete. I. Taxonomy,Production, Isolation and Biological Actmties

Abstract

Belactins A and B, new inhibitors of serine carboxypeptidase were discovered in the fermentation broth of Saccharopolyspora sp. MK19–42F6. They were purified by ethyl acetate extraction, silica gel chromatography, Sephadex LH20 chromatography, Capcellpak C18 SG120 reversed phase HPLC and centrifugal partition chromatography (CPC) following their inhibitory activity against carboxypeptidase Y (CP-Y). The inhibition constants (K_i) of belactins A and B against CP-Y are 0.14 and 0.27 μM respectively. Belactins A and B have highly specific inhibitory activities for CP-Y among various peptidases, have no antimicrobial activities at 100 μ/ml and have low toxicities.     

Characterization of a proton pump from pea stem microsomes

Abstract The present work deals with the characterization of an ATP-dependent proton translocation monitored by the ΔpH probe acridine orange. The ATP-dependent proton translocation has an optimum activity at pH 6.5 and is substrate specific for ATP. It is stimulated by Cl⁻, HCO₃⁻ and Br⁻, but is insensitive to several monovalent cations. Divalent cations (Mg²⁺ or Mn²⁺) are required for proton translocation, while in the presence of Ca²⁺ no uptake is observed. NO₃⁻, NO₂⁻ and citrate strongly inhibit proton uptake. On the contrary, F⁻, SO₄²⁻, malate, pyruvate, succinate, oxalate and acetate have no inhibitory effect. Proton uptake is stimulated by valinomycin and unaffected by molybdate. Two thiols, dithioerythritol and dithiothreitol, are able partially to prevent the FCCP-abolished proton uptake or partially restore the ATP-dependent proton translocation in FCCP-collapsed vesicles. It is suggested that pea stem microsomes possess an electrogenic ATPase, acting as a proton pump, which, on the basis of its characteristics, can be tentatively associated with membranes of tonoplast origin.     

Ethanol production by whole cell immobilization using lignocellulosic materials as solid matrix

Amongst four carriers used, rice-straw was found to be superior in terms of ethanol production. The maximum productivity (17.84 gl⁻¹ h⁻¹) corresponded to a dilution rate of 0.39 h⁻¹, the ethanol concentration being 45.80 gl⁻¹. A multistage rhomboidal bioreactor was found to partially overcome the disruption effect caused by the generation of a large volume of carbon dioxide in the column. Increases in productivity of about 12.55% and 3.6%, respectively, were achieved using rhomboidal and tapered bioreactors as compared to the cylindrical bioreactor. It was observed that the generation time of cells, in both the immobilized and free states, was around 2.5 h. The ethanol yield (Y_p/s) in the lower part of the reactor was less in comparison with other zones, where the substrate utilization efficiency was relatively higher.     

A new insight into lead (II) tolerance of environmental fungi based on a study of Aspergillus niger and Penicillium oxalicum

Environmental microorganisms have been widely applied in heavy metal remediation. This study explored the mechanisms of lead tolerance of two typical filamentous fungi, Aspergillus niger and Penicillium oxalicum. It is shown that the mechanisms of reducing Pb toxicity by these two fungi have three major pathways. The secreted oxalic acid can react with Pb (II) to form insoluble Pb minerals, primarily lead oxalate. Then, the enhanced biosorption via forming new border of cell wall prevents the transportation of Pb (II) into hypha. In addition, the fungal activity could be maintained even at high Pb concentration due to the intracellular accumulation. It was confirmed that A. niger has the higher Pb tolerance (up to 1500 mg l⁻¹ Pb level) compared with P. oxalicum (up to 1000 mg l⁻¹). Meanwhile, Pb levels below 1000 mg l⁻¹ partially stimulate the bioactivity of A. niger, which was confirmed by its elevated respiration (from 53 to 63 mg C l⁻¹ medium h⁻¹). This subsequently enhanced microbial functions of A. niger to resist Pb toxicity. A better understanding of Pb tolerance of these two fungi sheds a bright future of applying them to remediate lead-contaminated environments.