Separation and purification of l-methionine from E. coli fermentation broth by macroporous resin chromatography.

Methionine is an essential sulfur-containing amino acid for organisms. The separation and purification are important for the production of l-methionine from fermentation broth. In this work, the adsorption properties for l-methionine separation of ten macroporous resins were firstly evaluated. Macroporous cation resin D72 showed the best adsorption capacity (52.37 mg/g) and desorption rate (99.12%). The adsorption kinetics of l-methionine on D72 resin followed the pseudo second-order model and adsorption isotherm fitted well to the Sips model, the adsorption process mainly followed a physisorption mechanism. D72 resin packed columns were then used in the batch separation of fermentation broth, the operation parameters were optimized during the dynamic adsorption and desorption experiments. Under the optimized conditions: fermentation broth adjusted to pH = 2, loading flow rate at 2 BV/h, loading volume 55 mL, 1 mol/L NH3·H2O as eluent, elution flow rate at 2 BV/h, column height-diameter ratio at 14:1, excellent recovery and purity of l-methionine (82.37% and 85.69%, respectively) could be achieved.

[Separation of phenylalanine and methionine enantiomers by HPLC method: a comparison of stationary phase types]. / Separácia enantiomérov fenylalanínu a metionínu metódou HPLC: porovnanie typov stacionárnych fáz.

The paper deals with enantioselective separation of amino acids by the high performance liquid chromatography method. Separations of enantiomeric forms were tested on the chiral stationary phases based on ß-cyclodextrine, isopropyl carbamate cyclofructan 6, and the macrocyclic antibiotic teicoplanin. The best enantioseparation was obtained on the teicoplanin-based chiral stationary phase in the reversed-phase mode. UV spectrophotometric detection at 210 nm was used for detection of amino acids. The method was validated with respect to linearity, precision, limit of detection, limit of quantitation, and recovery. Limits of quantitation for phenylalanine and methionine enantiomers were 0.3 and 0.2 µg.ml-1, respectively. The HPLC method with teicoplanin-based chiral stationary phase was applied for analysis of dietary supplements.Key words: separation of enantiomers HPLC phenylalanine methionine.

Divergent functional roles of D-amino acids secreted by Vibrio cholera

The L-forms of amino acids are used in all kingdoms of life to synthesize proteins. However, the bacterium Vibrio cholerae, the causative agent of cholera, produces D-amino acids which are released to the environment at millimolar concentrations. We baptized these D-amino acids as non-canonical D-amino acids (NCDAAs) since they are different from those (i.e. D-alanine and D-glutamate) normally present in the bacterial cell wall. In V. cholerae, production of NCDAAs relies on the BsrV enzyme, a periplasmic broad spectrum racemase. BsrV multispecific activity, produces of a wide range of distinct D-amino acids. Using a combination of genetics and molecular physiology approaches we have demonstrated that NCDAAs target different cellular processes which may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that NCDAAs are relevant modulators of microbial subpopulations in diverse ecosystems (AU)

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Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide.

In cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions.

Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 µm totally porous silica particles of narrow size distribution.

A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2 µm totally porous silica particles of narrow size distribution. Columns of different lengths were packed at high pressure and a deep and systematic evaluation of kinetic performance, in terms of van Deemter analysis, was performed under different elution conditions (HILIC, POM, RP and NP) by using both achiral and chiral probes. For the achiral probes, the efficiency of the columns at the minimum of the van Deemter curves were very high leading to some 278,000, 270,000, 262,000 and 232,000 plates/m in hydrophilic interaction liquid chromatography (HILIC), polar organic mode (POM), normal phase (NP) and reversed phase (RP) respectively. The lowest plate height, Hmin=3.59 µm (h(/)=1.89), was obtained under HILIC conditions at a flow rate of 1.4 mL/min. Efficiency as high as 200,000-250,000 plates/m (at the optimum flow rate) was obtained in the separation of the enantiomers of chiral probes under HILIC/POM conditions. N-protected amino acids, α-aryloxy acids, herbicides, anti-inflammatory agents were baseline separated on short (2-cm) and ultra-short (1-cm) columns, with analysis time in the order of 1 min. The enantiomers of N-BOC-d,l-methionine were successfully baseline separated in only 11s in HILIC mode. Several examples of fast and efficient resolutions in sub/supercritical fluid chromatography were also obtained for a range of chiral carboxylic acids.

Penilumamide, a novel lumazine peptide isolated from the marine-derived fungus, Penicillium sp. CNL-338.

A novel lumazine peptide, penilumamide (1), was isolated from the fermentation broth of a marine-derived fungal strain, identified as Penicillium sp. (strain CNL-338) and the structure of the new metabolite was determined by analysis of ESI-TOF MS data combined with 1D and 2D NMR experiments.

Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers.

A new chiral stationary phase (CSP) containing thioester linkages was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to mercaptopropylsilica gel. The chiral recognition ability of the new CSP was found to be greater than that of the previously reported CSP containing amide linkages in the resolution of the various alpha-amino acids that were tested, except for that of Met, Ser and Thr. In the resolution of racemic amines and amino alcohols, the new CSP was always better than the one containing amide linkages in terms of the separation factors (alpha) and the resolutions (RS). Given the identical elution orders on the two CSPs, it was concluded that the chiral recognition mechanism is not affected by the change of the linkage type. In addition, the new CSP was found to be quite stable under the acidic mobile phase conditions that were utilized, indicating that the thioester linkage is useful as a tethering group.

[Effect of Panax ginseng components on the differentiation of mouse embryonic stem cells into cardiac-like cells].

Bioactive compounds that may control the specific differentiation from mouse embryonic stem (ES) cells into cardiac-like cells have been screened from herbal medicines. Among seven preparations, Panax ginseng was found to promote the differentiation into beating cells and to sustain their beating for longer than the control. Active compounds were found in its water-soluble fraction. Although they were not isolated, their candidates were surveyed in 42 compounds selected from the database of P. ginseng. Finally we found that vitamin B12 (VB12) and methionine were active. VB12 accelerated the differentiation into beating cells and made the beating rate constantly 100%. Moreover, VB12 was effective in the recovery of beating that was inhibited by spermine action. The mechanism of action of VB12 is discussed in termo of the relevance of intercellular electrical signal transduction.

Application of an eremomycin-chiral stationary phase for the separation of DL-methionine using simulated moving bed technology.

Recently a new chiral stationary phase (CSP) was introduced, based on the immobilization of the macrocyclic glycopeptide eremomycin to epoxy-activated silica. The application of this new CSP to preparative enantioseparation using simulated moving bed (SMB) chromatography will be presented. MeOH-H(2)O (0.1M NaH(2)PO(4))=20/80 (v/v) was used as the mobile phase to separate the enantiomers of methionine. Successful separation was realized providing productivities around 15 g(product)/l(stat)/h for both l and d-methionine under nonlinear conditions. In such delicate continuous chromatographic separation processes, besides productivity, the long-term stability of the applied stationary phases is of importance. Column to column fluctuations were negligible and long-term stability of the preparative stationary phase was satisfactory according to the results of perturbation experiments performed before and after long-term SMB runs.

Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions.

Silica-bonded teicoplanin aglycone allows enantioseparation of amino acids by reversed-phase liquid chromatography with a low organic solvent content. However, a reversible change in the adsorption behavior leading to a retention time shift (RTS) was observed when a preparative scale column was treated with harsh preparative chromatography-like conditions between finite-injection HPLC runs conducted under exactly the same conditions. This behavior was observed for all five investigated aliphatic and aromatic amino acids. In all cases, the retention times were prolonged after the overload conditions and the RTS was more pronounced for the later eluting d-enantiomer. We defined a standardized method for measuring the RTS and performed a systematic investigation on the influence of experimental conditions (type and concentration of pH modifier and organic modifier, temperature, pH) on the RTS. In this way a solvent composition--90/10 50 mM NH4Ac pH 5.8/MeOH--was identified that yielded no observable shift in retention time after overload conditions for both enantiomers. In order to treat the observed phenomenon on a mechanistic level, we applied band profile analysis based on the stochastic theory of chromatography and identified two different enantioselective sites. When the band profile analysis was performed on elution profiles obtained from runs with prolonged retention time after harsh overload conditions, the retention time shift could be attributed to both differentiable types of adsorption sites. One site was found to make both, enantioselective and non-selective contributions.

Analysis of methionine oxides and nitrogen-transporting amino acids in chilled and acclimated maize seedlings.

In maize seedlings, chilling causes a reduction of glutamine synthetase (GS) activity, while acclimation protects GS (manuscript submitted). Since ROS can oxidize both protein-bound and free Met to methionine sulfoxide (MSO) and further to methionine sulfone (MSO2, a GS inhibitor), it was hypothesized that the chilling-induced oxidative stress may cause accumulation of MSO and MSO2, thus contributing to the inactivation of GS. MSO2 preferentially inhibited the chloroplastic isoform, GS2. HPLC analysis of polar amino acids from coleoptiles + leaves, mesocotyls and roots of control, chilled, acclimated, acclimated and chilled and chilled and rewarmed plants revealed that free MSO and MSO2 do not accumulate after low temperature treatments. Nevertheless, acclimation significantly increased the expression of putative protein methionine sulfoxide reductase (PMSR), especially in mesocotyls. Different low temperature treatments caused complex changes in the profiles of N-transporting amino acids, Asp, Glu, Asn and Gln.

Cycling of volatile organic sulfur compounds in anaerobically digested biosolids and its implications for odors.

The objectives of this research were to elucidate the mechanisms for production and degradation of volatile organic sulfur compounds (VOSCs), key odor causing compounds produced by biosolids. These compounds included methanethiol (MT), dimethyl sulfide (DMS), and dimethyl disulfide (DMDS). A series of experiments were used to probe various pathways hypothesized to produce and degrade these VOSCs. The production of MT was found to mainly occur from degradation of methionine and the methylation of hydrogen sulfide. DMS was formed through the methylation of MT. DMDS was formed by MT oxidation. All three of the VOSCs were readily degraded by methanogens and a cyclic pathway was proposed to describe the production and degradation of VOSCs. The research demonstrated that the main source of VOSCs was the biodegradation of protein within the biosolids and the results provided a framework for understanding the production of odor from anaerobically digested sludges before and after dewatering.

An ionizable chromophoric reagent for the analysis of primary amine-containing drugs by capillary electrophoresis.

We found that ofloxacin acyl chloride is a potential chromophoric reagent for labeling amino analytes for capillary electrophoresis. Ofloxacin acyl chloride has a tertiary amino function in its structure and the derivatives from ofloxacin acyl chloride reacting with amino analytes can be ionized by an acid treatment and analyzed by simple capillary zone electrophoresis. Ofloxacin acyl chloride was used to derivatize model analytes (without chromophore) of amantadine (amino drug), tranexamic acid (non-protein amino carboxylic acid), glycine, and methionine (protein amino acids). The resulting derivatives were analyzed by capillary zone electrophoresis with ultraviolet detection (300 nm). The detection limits of the analytes studied were in the range of 1.0-2.5 microM (S/N = 3, injection 3 s). The precision (relative standard deviation) and accuracy (relative error) of the method for intra- and inter-day analyses of the analytes were respectively below 4.5% and 3.9%. Application of the method to the analysis of tranexamic acid in plasma proved feasible.

Methionine production by fermentation.

Fermentation processes have been developed for producing most of the essential amino acids. Methionine is one exception. Although microbial production of methionine has been attempted, no commercial bioproduction exists. Here, we discuss the prospects of producing methionine by fermentation. A detailed account is given of methionine biosynthesis and its regulation in some potential producer microorganisms. Problems associated with isolation of methionine overproducing strains are discussed. Approaches to selecting microorganism having relaxed and complex regulatory control mechanisms for methionine biosynthesis are examined. The importance of fermentation media composition and culture conditions for methionine production is assessed and methods for recovering methionine from fermentation broth are considered.

Fully automated synthesis module for preparation of S-(2-[(18)F]fluoroethyl)-L-methionine by direct nucleophilic exchange on a quaternary 4-aminopyridinium resin.

A fully automated preparation of S-(2-[(18)F]fluoroethyl)-L-methionine (FEMET), an amino acid tracer for tumor imaging with positron emission tomography, is described. [(18)F]F(-) was produced via nuclear reaction (18)O(p,n) [(18)F] at PETtrace Cyclotron. Direction nucleophilic fluorination reaction of [(18)F]fluoride with 1,2-di(4-methylphenylsulfonyloxy)ethane on a quaternary 4-(4-methylpiperidinyl)pyridinium functionalized polystyrene anion exchange resin gave 2-[(18)F]-1-(4-methylphenyl-sulfonyloxy)ethane, and then [(18)F]fluoroalkylation of L-homocysteine thiolactone with 2-[(18)F]-1-(4-methylphenylsulfonyloxy)ethane yielded FEMET. The overall radiochemical yield with no decay correction was about 10%, the whole synthesis time was about 52 min, and the radiochemical purity was above 95%.

Inventory and functional characterization of the HAK potassium transporters of rice.

Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters. To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake. We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

Immobilization of an L-aminoacylase-producing strain of Aspergillus oryzae into gelatin pellets and its application in the resolution of D,L-methionine.

The conditions for immobilization of an l-aminoacylase-producing strain of Aspergillus oryzae in gelatin and the enzymic characteristics of the immobilized pellets were studied. The optimal concentrations of gelatin, glutaraldehyde and ethyldiamine and time of immobilization were determined. Scanning electron micrographs reveal the cross-linked structure differences between the native and immobilized pellets. Optimum pH and temperature of the native and immobilized pellets were determined. Effects of ionic strength and substrate concentration on relative activity of the native and immobilized pellets were investigated in detail. The immobilized pellets were more stable over broader temperature and pH ranges. In addition, the immobilized pellets showed stable activity under operational and storage conditions. The immobilized pellets lost about 20% of their initial activity after five cycles of reuse. The results reported in this paper show the potential for using the immobilized A. oryzae pellets to resolve d,l-methionine.

Resolution of enantiomers of DL-amino acids on silica gel plates impregnated with optically pure (-)-quinine.

TLC resolution of enantiomers from racemic amino acids was achieved on silica gel plates impregnated with optically pure (-)-quinine. The successful solvent systems were butanol-chloroform-acetic acid (3:7:5, v/v) for DL-methionine; 6:8:4, v/v for alanine; 10:1:4; v/v for threonine; and ethyl acetate-carbon tetrachloride-propionic acid (10.5:6.5:3.5, v/v) for valine. Minimum detection limits were found to be different for each of the amino acid, ranging between 0.9 and 3.7 microg. The effects of concentration of impregnating reagent, temperature and pH on resolution of enantiomers have been studied in details.

High conversion in asymmetric hydrolysis during permeation through enzyme-multilayered porous hollow-fiber membranes.

We describe a novel porous hollow-fiber support for immobilizing aminoacylase in multilayers. Epoxy-group-containing polymer chains were grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization of glycidyl methacrylate, and subsequently a diethylamino group as an anion-exchange group was introduced into the graft chain. Aminoacylase was adsorbed in multilayers by allowing the amioacylase buffer solution to permeate through the pores across the hollow fiber; the graft chains provided three-dimensional space for the enzymes because of their electrostatic repulsion. The adsorbed enzyme at a degree of multilayer binding of 15 was cross-linked with glutaraldehyde to prevent leakage. An acetyl-DL-methionine solution was allowed to permeate through the pores surrounded by the aminoacylase-immobilized graft chain. Production of L-methionine was observed at a 4.1 mol/h per L of the fiber for a space velocity of 200 h(-1), defined as the flow rate of the effluent penetrating the outside surface of the hollow fiber divided by the membrane volume including the lumen.