Separation of saccharides using fullerene-bonded silica monolithic columns via π interactions in liquid chromatography.

We report on a potential method to separate sugars by using the specific interaction between fullerenes and saccharides in liquid chromatography (LC). Aromatic rings with high electron density are believed to interact strongly with saccharides due to CH-π and/or OH-π interactions. In this study, the fullerene-bonded columns were used to separate saccharides by LC under aqueous conditions. As a result, 2-aminobenzamide-labeled glucose homopolymer (Glcs) was effectively separated by both C60 and C70 columns in the range of Glc-1 to Glc-20 and high blood glucose level being retained in greater quantity. Furthermore, similar separations were identified by LC-mass spectrometry with non-labeled glucose homopolymers. Theoretical study based on molecular dynamics and DFT calculation demonstrated that a supramolecular complex of saccharide-fullerene was formed through CH-π and/or OH-π interactions, and that the interactions between saccharide and fullerene increase with the increase units of the saccharide. Additionally, the C60 column retained disaccharides containing maltose, trehalose, and sucrose. In this case, it was assumed that the retention rates were determined by the difference of the dipole moment in each saccharide. These results suggest that the dipole-induced dipole interaction was dominant, and that maltose-with the higher dipole moment-was more strongly retained compared to other disaccharides having lower dipole moment.

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

Phenylboronic acid modified solid-phase extraction column: Preparation, characterization, and application to the analysis of amino acids in sepia capsule by removing the maltose.

Maltose, a common auxiliary material of pharmaceutical preparation, may disturb the analysis of total amino acids in sepia capsule by aldolization. Therefore, it is necessary to remove the maltose through a convenient method. In this work, a phenylboronic acid modified solid-phase extraction column has been synthesized and used to remove the maltose. The materials were synthesized by one step "thiol-ene" reaction and the parameters of the column such as absorption capacity, recovery, and absorption specificity have been investigated. The results showed the column (0.5 cm of length × 0.5 cm of inner diameter) can absorb 4.6 mg maltose with a linear absorption and absorption specificity. Then this technique was applied in the quantification of amino acids in sepia capsule. After the optimization of the method, four kinds of amino acids, which were the most abundant, were quantified by high-performance liquid chromatography with diode array detection. The amounts of the four kinds of amino acids are 1.5∼2 times more than that without the treatment of solid-phase extraction column, which almost overcomes the influence of the maltose. All the results indicate that the phenylboronic acid modified solid-phase extraction column can successfully help to accurately quantify the total amino acids in sepia capsule.

Molecular structure, chemical properties and biological activities of Pinto bean pod polysaccharide.

Pinto bean pod polysaccharide (PBPP) was successfully extracted with yield of 38.5g/100g and the PBPP gave total carbohydrate and uronic acid contents of 286.2mg maltose equivalent/g and 374.3mgGal/g, respectively. The Mw of PBPP was 270.6kDa with intrinsic viscosity of 0.262dm(3)/g, which composed of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose (2.9%) and arabinose (4.3%) with trace amount of ribose and fucose. The result suggested that PBPP has a spherical conformation with a highly branched structure. Fourier Transform Infrared analysis showed that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film that was made of multiple micro strands of fibre. PBPP exhibited substantial free radical scavenging activity (7.7%), metal reducing capability (2.04mmol/dm(3)) and α-amylase inhibitory activity (97.6%) at a total amount of 1mg. PBPP also exhibited high water- and oil-holding capacities (3.6g/g and 2.8g/g, respectively). At a low concentration, PBPP exhibited emulsifying activity of 39.6% with stability of 38.6%. Apart from that, PBPP was able to show thickening capability at low concentration (0.005kg/dm(3)).

Particulate structure of phytoglycogen studied using ß-amylolysis.

Phytoglycogen (PG), a dendrimer-like glucan particulate, has a much higher dispersed molecular density than amylopectin (AP). In this study, ß-amylase was used to investigate the effect of high molecular density of PG on its susceptibility to enzymatic hydrolysis. AP and PG reached the limit of ß-amylolysis at 20 and 480 min, respectively, suggesting a much higher resistance of PG to ß-amylase. The majority of PG ß-amylolysis occurred in the initial 2 min, followed by a slow progression that implied low accessibility of internal particulate portion to enzyme. The chain length profile of PG ß-limit dextrin showed only one population of long chains, indicating the absence of branch clusters with PG. At the limit of ß-amylolysis, a substantial decrease in the molar mass was observed for both PG and AP, whereas only a slight reduction in the Z-average root mean square radius was observed for PG (from 24.5 to 23.1 nm) compared to that of AP (from 91.1 to 69.6 nm).

Kinetics and equilibria of the chromatographic separation of maltose and trehalose.

Trehalose, a nonreducing disaccharide, has been extensively applied to food, cosmetics, and pharmaceutical goods. The resultant solution of trehalose prepared by enzymatic methods includes high amounts of maltose. However, it is quite difficult to separate maltose and trehalose on an industrial scale because of their similar properties. In this paper, a high-performance resin was selected as a stationary phase to separate trehalose and maltose, and the resolution of these sugars was 0.59. The potential of a cation exchange resin was investigated as the stationary phase in separating trehalose and maltose using deionized water as the mobile phase. Based on the equilibrium dispersive model, the axial dispersion coefficients and overall mass transfer coefficients of maltose and trehalose were determined by moment analysis at two different temperatures, 50 and 70°C. Other parameters, including the column void and the adsorption isotherms, were also determined and applied to simulate the elution curves of trehalose and maltose. The simulated results matched the experimental data, validating the parameters. The optimized parameters are critical to the chromatographic separation of trehalose and maltose on an industrial scale.

Overexpression, purification and biophysical characterisation of E. coli MerT.

Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg(2+) into cytoplasm and enzymic reduction to volatile Hg(0). MerT is a small (13kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-ß-d-maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering.

Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304.

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K m and V max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.

Specificity of maltase to maltose in three different directions of reaction: hydrolytic, vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis.

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K(m) value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate.

Lipase-catalyzed selective synthesis of monolauroyl maltose using continuous stirred tank reactor.

Monolauroyl maltose was synthesized by an immobilized lipase that catalyzed condensation of maltose and lauric acid in acetone using a batch reactor or a continuous stirred tank reactor. Mono- and di-lauroyl maltoses were identified by FT-IR, (1)H NMR, (13)C NMR and MS. Monolauroyl maltose was selectively synthesized in a continuous stirred tank reactor and no diester was detected. The highest concentration of monolauroyl maltose at 28 mmol/l was obtained in 250 ml acetone when maltose was added at 4 g/d and the molar ratio of lauric acid to maltose was fixed at 4:1 at a flow rate of 0.15 ml/min for both influx and effluent without supplement of fresh molecular sieve.

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration.

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins.

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.

[Separation of saccharides in tobacco by capillary zone electrophoresis].

A method for the separation of derivatized saccharides in tobacco with high performance capillary electrophoresis and ultraviolet spectrophotometry (HPCE-UV) is presented. Five saccharides in tobacco were derivatized with p-aminobenzonitrile, separated at pH 10.5 with 50 mmol/L borate buffer containing 5% (volume fraction, the same in the following percentage concentration) methanol, 5% acetonitrile, 2.5% ethyleneglycol, 2.5% 2-propanol and 1 mmol/L sodium dodecyl sulfate, and detected at 285 nm.

Expression of an active recombinant lysine 49 phospholipase A(2) myotoxin as a fusion protein in bacteria.

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.

T-Cell reactivity to the P2C nonstructural protein of a diabetogenic strain of coxsackievirus B4.

Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP-P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-gamma or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.

Expression and purification of recombinant proteins by fusion to maltose-binding protein.

The pMAL vectors provide a method for purifying proteins from cloned genes by fusing them to maltose-binding protein (MBP, product of malE), which binds to amylose. The vectors use the tac promoter and the translation initiation signals of MBP to give high-level expression of the fusion, and an affinity purification for MBP to isolate the fusion protein. The pMAL polylinkers carry restriction sites to insert the gene of interest, and encode a site for a specific protease to separate MBP from the target protein after purification. Vectors with or without the malE signal sequence can be used, to express the protein cytoplasmically for the highest level of production or periplasmically to help in proper folding of disulfide-bonded proteins.

Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from [14C] maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase.

Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism. 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues. The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase. The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K. oxytoca M5a1.

Exploitation of a monoclonal antibody for weak affinity-based separation in capillary gel electrophoresis.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

TLC separation of some common sugars on silica gel plates impregnated with transition metal ions.

TLC separation of glucose, maltose, lactose, sorbitol and sucrose on silica gel plates impregnated with transition metal ions, Cu(II), NI(II), Zn(II) or Cd(II), has been achieved. The identification is very distinct by using KMnO4 (0.5%) in 0.1 M NaOH as the spray reagent. Two new solvent systems, (A) n-PrOH-H2O) (8:4, v/v), and (B) i-PrOH-H2O (8:4, v/v), were worked out.