Production of ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. using resin-based,in situ product removal

Resin-based, in situ product removal (ISPR) was used to increase production of ε-poly-l-lysine (PL) by Streptomyces sp. GIM8. D152 resin was selected over Amberlite IRC-50, Amberlite IRC-76 and Amberlite IR-120 to develop ISPR using adsorption capacity and desorption ratio as bases. The yield of PL in response to external PL was unaffected in shake-flask culture; however, the production of PL increased to 2.9 from 0.8 g l⁻¹ shake-flasks using ISPR. In a 5 l fermentor, 23.4 g PL l⁻¹ was achieved compared to 3.76 g PL l⁻¹, in the controls by attaching two bags of D152 resin to the probes and baffles of the fermentor.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Iron(III) complexation by Vanchrobactin, a siderophore of the bacterial fish pathogen Vibrio anguillarum

The bacterial fish pathogen Vibrio anguillarum serotype O2 strain RV22 produces the mono catecholate siderophore Vanchrobactin (Vb) under conditions of iron deficiency. Vb contains two potential bidentate coordination sites: catecholate and salicylate groups. The iron(III) coordination properties of Vb is investigated in aqueous solutions using spectrophotometric and potentiometric methods. The stepwise equilibrium constants (log?K) for successive addition of Vb dianion to a ferric ion are 19.9; 13.3, and 9.5, respectively, for an overall association constant of 42.7. Based on the previous results, we estimated the equilibrium concentration of free iron(III) under physiological conditions for pH 7.4 solution containing 10(-6) M total iron and 10(-5) M total Vb as pFe = 20 (=-log[Fe(3+)]). The Vb model compounds catechol (Cat) and 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide (Dhb) have also been examined, and the obtained results show that the interaction of the whole system of Vb that contains the ferric-chelating groups of both Dhb and Cat, is synergically greater than the separate parts; i.e. Vb is the best chelating agent either in acid or basic media. In summary, bacteria employing Vb-mediated iron transport thus are able to compete effectively for iron with other microorganisms within which they live.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Solid phase synthesis of peptide hydroxamic acids on poly(ethylene glycol)‐based support

A novel resin designed for solid‐phase synthesis of peptide hydroxamic acids (PHA) combining the trityl linker with poly(ethylene glycol)‐based support, ChemMatrix® type, is described. The synthesis of PHA can be performed according to a standard protocol, providing products in excellent purity and reasonable yields. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Complexation of labile aluminium species by chelating resins Iontosorb--a new method for Al environmental risk assessment

The utilization of chelating ion-exchange by the method based on binding strength and kinetic discrimination for aluminium fractionation was studied. Two chelating cellulose resins, Iontosorb Oxin (IO) and Iontosorb Salicyl (IS), were used for the determination of quickly reacting labile aluminium species. The possibilities of aluminium fractionation on these chelating resins were investigated by a solid phase extraction technique. The study of the pH (2.5-6.0) influence on the Al complexation by both resins indicates that at low pH the IS has lower sorption capacity but better adsorptive kinetic properties than IO. The optimal resin complexation time for reactive Al species was experimentally found after aluminium sorption study at pH 4.0 in synthetic solutions containing some inorganic and organic ligands, which simulate the composition of analysed acid soil and water samples. The negative influence of sulphate and iron on the Al complexation by IS resin was found and investigated. The flame atomic absorption spectrometry was used for the aluminium quantification.     

Estimation of catecholamines in human plasma by ion-exchange chromatography coupled with fluorimetry

Estimation of catecholamines in human plasma was made by ion-exchange chromatography coupled with fluorimetry.Catecholamines in deproteinized plasma were adsorbed onto Amberlite CG-50 (pH 6.5, buffered with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid. The catecholamine fraction was separated further on a column of Amberlite IRC-50 which was coupled with a device for the automated performance of the trihydroxyindole method (epinephrine and norepinephrine) or the 4-aminobenzoic acid—oxidation method (dopamine). One sample could be analysed within 25 min with either method. The lower detection limits were 0.02 ng for epinephrine and dopamine, and 0.04 ng for norepinephrine.Plasma catecholamine contents of healthy adults at rest were epinephrine 0.07 ± 0.01 ng/ml (n = 19), norepinephrine 0.27 ± 0.03 ng/ml (n = 19) and dopamine 0.22 ± 0.03 ng/ml (n = 26).The procedure of adsorption and elution of the plasma catecholamines by ion-exchange resin was simple, the simplicity contributing to constant recovery. The catecholamine fraction could be analysed without evaporation of the eluate. The analytical column could be used for the analysis of more than 1000 samples before excessive back-pressure developed. Our method of continuous measurement of plasma catecholamine fulfils clinical requirements.     

Studies on Metabolic Pathway of NAD in Yeast Cells

A new method for preparing NMN (nicotinamide mononucleotide) by the use of yeast 5′-nucleotidase is presented. After hydrolysis of NAD into NMN, adenosine and P_i by yeast 5′-nucleotidase which is a single protein having nucleotide pyrophosphatase activity, NMN in the hydrolysate of NAD was purified on active carbon and subsequently on Amberlite IRC-50.

In the typical experiment, 0.74 g of NMN (88% purity) was obtained from 2g of NAD preparation, giving 76% recovery on the basis of the theoretical value.

The NMN preparation was identified as NMN by IR spectra, UV spectra, paper chromatography, and also by component analysis.     

Purification of poly-dA oligonucleotides and mRNA-protein fusions with dT25-OAS resin

Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT₂₅-conjugated Oligo Affinity Support resin (dT₂₅-OAS) alongside poly-dT₁₄ magnetic beads and dT₂₅-cellulose. dT₂₅-OAS was found to have the highest dA₂₁ oligo binding capacity at 4 pmol/µg, followed by dT₁₄-magnetic beads (1.1 pmol/µg) and dT₂₅-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT₂₅-cellulose showed the highest mRNA-affibody purification specificity, followed by dT₂₅-OAS and dT₁₄-magnetic beads. Overall, dT₂₅-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.     

Voltammetric determination of dissolved iron and its speciation in freshwater

Analytical methods were developed to determine the concentration of total dissolved iron and its chemical speciation in freshwater using cathodic stripping voltammetry (CSV) with 1-nitroso-2-naphthol (NN) at pH 8.1. The concentrations of total dissolved iron in river water that iron concentration was certified and in natural water samples from Lake Kasumigaura were determined successfully. The natural iron ligand concentration and the conditional stability constant were determined by ligand competition between NN and the natural ligands present in the sample. In the water samples from Lake Kasumigaura, the concentrations of total dissolved iron and natural ligand were 47.8 ± 4.4nM and 80.0 ± 19.6nM and the conditional stability constant (K_FeL) was 10^25.9±0.4M^–1 (n = 3). The value of K_FeL was greater than any reported K_FeL for seawater. More than 99.9% of the dissolved iron existed as organic species due to the very high value of the conditional stability constant. The inorganic iron concentration calculated from these results was 10^–13.4M, indicating that the inorganic iron level in Lake Kasumigaura was similar to that in the open ocean and therefore that iron can be a limiting factor for algal growth in Lake Kasumigaura. This is the first report of the complexation of iron(III) and inorganic iron levels in lake water determined by CSV.     

Stereoselective catalysis by Fe(III) complex ions supported on asymmetric polymers: Oxidation of ascorbic acid in aqueous solution

Quaterpyridyneiron (III) complex ions anchored to partially ordered poly (L-glutamate) or poly (D-glutamate) were used as (enantiomeric) catalysts for the H₂O₂-oxidation of L(+) ascorbic acid at pH 7. When the α-helical fraction of polypeptide matrices was low, the configuration dissymmetry of the active sites was unable to impart any stereoselective effect in the catalysis, i.e. k = 3.66 x 10³ M^?1?sec^?1 (25.9°C) with both catalysts. On the contrary, by increasing the amount of α-helix in the polymeric supports the stereoselectivity increases, the second-order rate constants k_FeD being definitely higher than k_FeL.Implications of the role played by the conformational dissymmetry of the active sites in the stereospecificity of the process are briefly discussed.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Synthesis of spherical Fe2O3 nanoparticles from the thermal decomposition of iron (III) nano-structure complex: DFT studies and evaluation of the biological activity

A novel Fe(III) Schiff base complex of the [FeL₂(NO₃)₂]NO₃ type where L = 2-((pyridin-4-yl)methyleneamino)-3-aminomaleonitrile was synthesized using the reflux and sonochemical methods and their antibacterial and antifungal activity were evaluated. The nanoparticles of iron oxide (Fe₂O₃) were obtained from the iron nano-structure complex as a precursor after calcination at 600 ˚C for 3 h. All the synthesized compounds were characterized by various spectroscopic techniques. The results of SEM showed that the morphology of iron nano-structure complex was rod-like while the morphology of the Fe₂O₃ nano powder was spherical. The results of the biological studies indicated that the iron nano-structure complex showed a stronger antibacterial and antifungal efficiency than its bulk complex. Finally, the empirical geometrical parameters of complexes revealed a good agreement with calculated ones at DFT-B3LYP level.     

Partition coefficients (free ligands and their iron(III) complexes) and lipophilic behavior of new abiotic chelators. Correlation to biological activity

Partition coefficients between n-octanol and water have been measured for ten tripodal ligands with catecholate or hydroxyquinolinate or pyridinophenolate chelating subunits and for their iron(III) complexes. The abilities of the ligands to cross an octanol phase and to extract ferric ion from its EDTA complex in an aqueous phase are studied. Correlation with biological properties are discussed.     

Trace metal complexation by the triscatecholate siderophore protochelin: structure and stability

Although siderophores are generally viewed as biological iron uptake agents, recent evidence has shown that they may play significant roles in the biogeochemical cycling and biological uptake of other metals. One such siderophore that is produced by A. vinelandii is the triscatecholate protochelin. In this study, we probe the solution chemistry of protochelin and its complexes with environmentally relevant trace metals to better understand its effect on metal uptake and cycling. Protochelin exhibits low solubility below pH 7.5 and degrades gradually in solution. Electrochemical measurements of protochelin and metal–protochelin complexes reveal a ligand half-wave potential of 200 mV. The Fe(III)Proto³⁻ complex exhibits a salicylate shift in coordination mode at circumneutral to acidic pH. Coordination of Mn(II) by protochelin above pH 8.0 promotes gradual air oxidation of the metal center to Mn(III), which accelerates at higher pH values. The Mn(III)Proto³⁻ complex was found to have a stability constant of log β₁₁₀ = 41.6. Structural parameters derived from spectroscopic measurements and quantum mechanical calculations provide insights into the stability of the Fe(III)Proto³⁻, Fe(III)H₃Proto, and Mn(III)Proto³⁻ complexes. Complexation of Co(II) by protochelin results in redox cycling of Co, accompanied by accelerated degradation of the ligand at all solution pH values. These results are discussed in terms of the role of catecholate siderophores in environmental trace metal cycling and intracellular metal release.     

Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.     

Production of S-adenosyl-l-methionine by Saccharomyces sake

In a study of the optimization of the culture conditions for the intracellular accumulation of S-adenosyl-l-methionine (AdoMet) by Saccharomyces sake Kyokai No. 6, a medium containing 10% sucrose, 1.8% urea, 1% yeast extract and 0.75–1.5% l-methionine as the main components was found to be suitable for the production. Addition of CaCl₂, biotin and glycylglycine to the medium increased the accumulation of AdoMet. Under optimal conditions in a 10-liter fermenter, the yeast produced 10.8 g L⁻¹ AdoMet (260 mg per g dry cells).In order to increase the recovery yield and purity of AdoMet, a new and efficient isolation procedure involving freezing and thawing of cells for the extraction of AdoMet and chromatographies on Amberlite IRA-45, XAD-2 and IRC-50 was developed.     

Hydrolysis of methyloleate using immobilized lipase from Chromobacterium viscosum

Various lipases were screened for their hydrolytic efficiency towards methyloleate. Lipase from Chromobacterium viscosum gave highest hydrolysis efficiency of 92% in 24?h. Different cation exchange resins were screened to immobilize lipase from Chromobacterium viscosum. A weakly acidic macroreticular type resin, IRC-50 having carboxyl end group functionality gave highest activity yield of 18.8%. Strongly acidic cation exchange resins with sulphonic functionality and macroreticular type did not give much activity yield when compared to weakly acidic non macroreticular type resins. It was observed that end group functionality and structure of the matrices plays an important role in obtaining highest activity yield. For a specific water concentration, the hydrolysis ratio reached 85% in less than 7?h when the substrate to enzyme ratio was 4. As the ratio is increased above 4, the availability of water at the interface has become a limitation for obtaining maximum hydrolysis.