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Covalent Binding (covalent + binding)
Selected AbstractsNovel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activityCONTRAST MEDIA & MOLECULAR IMAGING, Issue 4 2010Lorenzo Tei Abstract Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd,DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material. Copyright © 2010 John Wiley & Sons, Ltd. [source] Slow desorption behavior of one highly resistant aromatic amine in Lake Macatawa, Michigan, USA, sedimentENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2005Shihua Chen Abstract The desorption behavior of benzidine from Lake Macatawa (Holland, MI, USA) sediment was investigated in this study using batch solvent extraction method. Seven solvents were tested as the extracting reagents: Deionized water (DI), calcium chloride in DI (CaCl2), sodium hydroxide in DI (NaOH), acetonitrile (ACN), a mixture of acetonitrile and ammonium acetate in DI (ACNNH4OAc), methanol (MeOH), and hydrochloric acid in DI (HCl). These solvents are proposed to react with sediment-associated benzidine by different mechanisms (e.g., cation exchange, hydrophobic partitioning, and covalent binding). Three sets of sorption isotherm experiments were conducted separately in these seven solvents with a 7-d, three-week, and two-month contact time. The results demonstrated nonlinear isotherms with Freundlich 1/n values varying from 0.25 to 0.52. The desorption behavior of benzidine in the solvents was evaluated after the sorption of benzidine onto the sediment with same contact times of 7 d, three weeks, and two months. A two-stage model subsequently was applied to simulate the experimental data. The rapidly desorbing rate constants were on the order of one to two per day for ACN, ACN-NH4OAc, and NaOH solvents, and the slowly desorbing rate constants were on the order of 10,5 to 10,4/d. Sequential desorption experiment demonstrated low total extraction efficiency of less than 40%. Both the observed sorption and desorption phenomena suggested that hysteresis and/or mass-transfer limited diffusion may result in the slow desorption behavior observed in this study. [source] Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophagesFEBS JOURNAL, Issue 6 2007Bronwyn E. Brown Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glucose or metabolic processes) to low-density lipoproteins has been previously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atherosclerosis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been characterized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester levels were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was significantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Differences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumulation of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls. [source] Kinetics and thermodynamics of nick sealing by T4 DNA ligaseFEBS JOURNAL, Issue 21 2003Alexey V. Cherepanov T4 DNA ligase is an Mg2+ -dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a ,processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a ,nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (< 10 µm) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (, 0.2 min,1) and a subsequent slow ligation (, 2 × 10,3 min,1). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal·mol,1), transadenylation of the nick (0.9 kcal·mol,1), and nick-sealing (16.3,18.8 kcal·mol,1) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase,AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis. [source] Selective Zinc(II)-Ion Fluorescence Sensing by a Functionalized Mesoporous Material Covalently Grafted with a Fluorescent Chromophore and Consequent Biological ApplicationsADVANCED FUNCTIONAL MATERIALS, Issue 2 2009Krishanu Sarkar Abstract A highly ordered 2D-hexagonal mesoporous silica material is functionalized with 3-aminopropyltriethoxysilane. This organically modified mesoporous material is grafted with a dialdehyde fluorescent chromophore, 4-methyl-2,6-diformyl phenol. Powder X-ray diffraction, transmission electron microscopy, N2 sorption, Fourier transform infrared spectroscopy, and UV-visible absorption and emission have been employed to characterize the material. This material shows excellent selective Zn2+ sensing, which is due to the fluorophore moiety present at its surface. Fluorescence measurements reveal that the emission intensity of the Zn2+ -bound mesoporous material increases significantly upon addition of various concentrations of Zn2+, while the introduction of other biologically relevant (Ca2+, Mg2+, Na+, and K+) and environmentally hazardous transition-metal ions results in either unchanged or weakened intensity. The enhancement of fluorescence is attributed to the strong covalent binding of Zn2+, evident from the large binding constant value (0.87,×,104M,1). Thus, this functionalized mesoporous material grafted with the fluorescent chromophore could monitor or recognize Zn2+ from a mixture of ions that contains Zn2+ even in trace amounts and can be considered as a selective fluorescent probe. We have examined the application of this mesoporous zinc(II) sensor to cultured living cells (A375 human melanoma and human cervical cancer cell, HeLa) by fluorescence microscopy. [source] Selective Immobilization of Protein Clusters on Polymeric Nanocraters,ADVANCED FUNCTIONAL MATERIALS, Issue 9 2006A. Valsesia Abstract A method for fabricating chemically nanopatterned surfaces based on a combination of colloidal lithography and plasma- enhanced chemical vapor deposition (PECVD) is presented. This method can be applied for the creation of different nanopatterns, and it is in principle not limited in patterning resolution. Nanocraters of poly(acrylic acid) (carboxylic moieties) surrounded by a matrix of poly(ethylene glycol) are fabricated. Chemical force microscopy demonstrates that the process is able to produce the expected surface chemical contrast. Finally, the carboxylic groups of the craters are activated in order to induce the covalent binding of fluorescent-labeled proteins. Fluorescence investigation using scanning confocal microscopy shows that the proteins are preferentially attached inside the functional craters. [source] Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liverHEPATOLOGY, Issue 2 2002Ping Zhao Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N -acetyl- p -benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione- S -transferase , released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by ,2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were ,50% more susceptible to NAPQI (25-165 ,mol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH. [source] Fabrication of Oriented Multilayers of Photosystem I Proteins on Solid Surfaces by Auto-Metallization,ADVANCED MATERIALS, Issue 2 2008L. Frolov AFM phase image of PS I (Top) and platinized PS I (bottom) monolayers. The Pt patches serve as junctions for fabrication of oriented multilayer by covalent binding of cysteine mutants through the formation of sulfide bonds with the Pt. A larger absorption cross section and the serial arrangement resulted in an increase in the photopotential generated by the dry multilayers. [source] Determinants of skin sensitisation potentialJOURNAL OF APPLIED TOXICOLOGY, Issue 3 2008David W. Roberts Abstract Skin sensitisation is an important toxicological endpoint. The possibility that chemicals used in the workplace and in consumer products might cause skin sensitisation is a major concern for individuals, for employers and for marketing. In European REACH (Registration, Evaluation, and Authorisation of Chemicals) legislation, the sensitising potential should therefore be assessed for chemicals below the 10 ton threshold. Development of methods for prediction of skin sensitisation potential without animal testing has been an active research area for some time, but has received further impetus with the advent of REACH and the EU Cosmetics Directive (EU 2003). This paper addresses the issue of non-animal based prediction of sensitisation by a mechanistic approach. It is known that the sequence of molecular, biomolecular and cellular events between exposure to a skin sensitiser and development of the sensitised state involves several stages, in particular penetration through the stratum corneum, covalent binding to carrier protein, migration of Langerhans cells, presentation of the antigen to naďve T-cells. In this paper each of these stages is considered with respect to the extent to which it is dependent on the chemical properties of the sensitiser. The evidence suggests that, although penetration of the stratum corneum, stimulation of migration and maturation of Langerhans cells, and antigen recognition are important events in the induction of sensitisation, except in certain specific circumstances they can be taken for granted. They are not important factors in determining whether a compound will be a sensitiser or not, nor are they important factors in determining how potent one sensitiser will be relative to another. The ability to bind covalently to carrier protein is the major structure-dependent determinant of skin sensitisation potential. A chemistry-based prediction strategy is proposed involving reaction mechanistic domain assignment, reactivity and hydrophobicity determination, and application of quantitative mechanistic modelling (QMM) or read-across. Copyright © 2007 John Wiley & Sons, Ltd. [source] Inhibition of human squalene monooxygenase by selenium compoundsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2002Nisha Gupta Abstract Selenosis in animals is characterized by a variety of neurological abnormalities, but the chemical species of selenium and the molecular targets that mediate this neurotoxicity are unknown. We have previously shown that selenite is a potent inhibitor of squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis; inhibition of this enzyme by dimethyltellurium leads to a peripheral demyelinating neuropathy similar to that seen in selenosis. To evaluate the role methylation plays in selenium toxicity, we examined the ability of three methylselenium compounds, methylselenol, dimethylselenide, and trimethylselenonium iodide, to inhibit purified recombinant human squalene monooxygenase. IC50 values for methylselenol (95 ,M) and dimethylselenide (680 ,M) were greater than that previously obtained for selenite (37 ,M), and inhibition by trimethylselenonium iodide was evident only at concentrations above 3 mM. Inhibition by methylselenol as well as by selenite was slow and irreversible, suggestive of covalent binding to the enzyme, and thiol-containing compounds could prevent and reverse this inhibition, indicating that these compounds were reacting with sulfhydryl groups on the protein. Monothiols such as glutathione and ,-mercaptoethanol provided better protection than did dithiols, suggesting that these selenium compounds bind to only one of the two proposed vicinal cysteines on squalene monooxygenase. Unexpectedly, the inhibition by selenite was significantly enhanced by dithiols, indicating that a more toxic species, possibly selenide, was formed in the presence of these dithiol reductants. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:18,23, 2002; DOI 10.1002/jbt.10014 [source] Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilizationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2001Jesús Torres-Bacete Abstract Penicillin,V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy-activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55,°C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10-fold increase in its half-life at 40,°C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry [source] Adsorption Kinetics and Thermodynamics of Acid Dyes on a Carboxymethylated Chitosan-Conjugated Magnetic Nano-AdsorbentMACROMOLECULAR BIOSCIENCE, Issue 3 2005Yang-Chuang Chang Abstract Summary: The monodisperse chitosan-conjugated Fe3O4 nanoparticles with a mean diameter of 13.5 nm were fabricated by the carboxymethylation of chitosan and its covalent binding onto Fe3O4 nanoparticles via carbodiimide activation. The carboxymethylated chitosan (CMCH)-conjugated Fe3O4 nanoparticles with about 4.92 wt.-% of CMCH had an isoelectric point of 5.95 and were shown to be quite efficient as anionic magnetic nano-adsorbent for the removal of acid dyes. Both the adsorption capacities of crocein orange G (AO12) and acid green 25 (AG25), as the model compounds, decreased with increasing pH, and the decreasing effect was more significant for AO12. On the contrary, the increase in the ionic strength decreased the adsorption capacity of AG25 but did not affect, obviously, the adsorption capacity of AO12. By the addition of NaCl and NaOH, both AO12 and AG25 could desorb and their different desorption behavior could be attributed to the combined effect of pH and ionic strength. From the adsorption kinetics and thermodynamics studies, it was found that both the adsorption processes of AO12 and AG25 obeyed the pseudo-second-order kinetic model, Langmuir isotherm, and might be surface reaction-controlled. Furthermore, the time required to reach the equilibrium for each one was significantly shorter than those using the micro-sized adsorbents due to the large available surface area. Also, based on the weight of chitosan, the maximum adsorption capacities were 1,883 and 1,471 mg,·,g,1 for AO12 and AG25, respectively, much higher than the reported data. Thus, the anionic magnetic nano-adsorbent could not only be magnetically manipulated but also possessed the advantages of fast adsorption rate and high adsorption capacity. This could be useful in the fields of separation and magnetic carriers. Acid dyes adsorption onto the CMCH-conjugated Fe3O4 nanoparticles. [source] Binding and Sulfonation of Poly(acrylic acid) on Iron Oxide Nanoparticles: a Novel, Magnetic, Strong Acid Cation Nano-AdsorbentMACROMOLECULAR RAPID COMMUNICATIONS, Issue 19 2005Sou-Yee Mak Abstract Summary: A novel, magnetic, strong acid cation nano-adsorbent has been developed by the covalent binding of poly(acrylic acid) on the surface of Fe3O4 nanoparticles followed by sulfonation using sulfanilic acid via carbodiimide activation. The nano-absorbent can be easily recovered or manipulated with an external magnetic field and shows a good capacity for the rapid and efficient adsorption of multivalent metal cations from aqueous solutions. An illustration for the binding and sulfonation of PAA on Fe3O4 nanoparticles to produce a magnetic, strong acid cation nano-adsorbent. [source] Random Computer Generation of 3D Molecular Structures: Theoretical and Statistical AnalysisMACROMOLECULAR THEORY AND SIMULATIONS, Issue 2 2006Alain Porquet Abstract Summary: A computer program has been developed to generate three-dimensional molecular structures randomly from a given collection of elementary chemical functional groups: the so-called fragment database. The gradual assembly of the various fragments present in the database is performed according to a "self-generation algorithm" (SGA). The latter is based on the covalent binding, step by step, between the unoccupied electronic valencies associated with the fragments of the database, and those of the growing molecular structure. When the number of electronic valencies of the molecular structure is zero, the growth process for this particular molecule is completed. It is shown that SGA is able to reproduce the asymmetric mass distributions of some natural colloids, like humic substances. In this article, particular attention is given to the analysis of the relationship existing between the fragment composition of the database and that of the collection of molecules generated. Mathematical expressions are derived and discussed, to understand the relationship between the proportions of the different types of fragments and the final composition of the generated molecular ensembles. For that purpose, a "pathway" formalism is proposed to describe exhaustively the whole set of generated molecules by specifying the distribution function of all of the fragments therein integrated. A statistical analysis that satisfactorily reproduces the predictions of the pathway model is extensively discussed. Example of a three-dimensional structure obtained with the "self-generation algorithm" (SGA). [source] Techniques for phosphopeptide enrichment prior to analysis by mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 1 2010Jamie D. Dunn Abstract Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29,54, 2010 [source] Production, characterization and determination of the real catalytic properties of the putative ,succinate dehydrogenase' from Wolinella succinogenesMOLECULAR MICROBIOLOGY, Issue 5 2009Hanno D. Juhnke Summary Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as ,non-classical' succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86,His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions. [source] Improved detection of reactive metabolites with a bromine-containing glutathione analog using mass defect and isotope pattern matchingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2010André LeBlanc Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using invitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N -(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the invitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. Invitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd. [source] Immobilization of a thermostable ,-amylase by covalent binding to an alginate matrix increases high temperature usabilityBIOTECHNOLOGY PROGRESS, Issue 2 2009Boon L. Tee Abstract Thermostable ,-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized ,-amylase were investigated. Covalently bound thermostable ,-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound ,-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (Km) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (Vmax) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable ,-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] High Production of D -Tagatose, a Potential Sugar Substitute, Using Immobilized L -Arabinose IsomeraseBIOTECHNOLOGY PROGRESS, Issue 1 2001Pil Kim An L -arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D -tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L -arabinose isomerase stably produced an average of 7.5 g-tagatose/L·day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U·day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D -Tagatose production using an immobilized L -arabinose isomerase has a high potential for commercial application. [source] Covalent Attachment of Bacteriorhodopsin Monolayer to Bromo-terminated Solid Supports: Preparation, Characterization, and Protein StabilityCHEMISTRY - AN ASIAN JOURNAL, Issue 7 2008Yongdong Jin Dr. Abstract The interfacing of functional proteins with solid supports and the study of related protein-adsorption behavior are promising and important for potential device applications. In this study, we describe the preparation of bacteriorhodopsin (bR) monolayers on Br-terminated solid supports through covalent attachment. The bonding, by chemical reaction of the exposed free amine groups of bR with the pendant Br group of the chemically modified solid surface, was confirmed both by negative AFM results obtained when acetylated bR (instead of native bR) was used as a control and by weak bands observed at around 1610,cm,1 in the FTIR spectrum. The coverage of the resultant bR monolayer was significantly increased by changing the pH of the purple-membrane suspension from 9.2 to 6.8. Although bR, which is an exceptionally stable protein, showed a pronounced loss of its photoactivity in these bR monolayers, it retained full photoactivity after covalent binding to Br-terminated alkyls in solution. Several characterization methods, including atomic force microscopy (AFM), contact potential difference (CPD) measurements, and UV/Vis and Fourier transform infrared (FTIR) spectroscopy, verified that these bR monolayers behaved significantly different from native bR. Current,voltage (I,V) measurements (and optical absorption spectroscopy) suggest that the retinal chromophore is probably still present in the protein, whereas the UV/Vis spectrum suggests that it lacks the characteristic covalent protonated Schiff base linkage. This finding sheds light on the unique interactions of biomolecules with solid surfaces and may be significant for the design of protein-containing device structures. [source] Organometallic Ruthenium Inhibitors of Glutathione- S -Transferase P1-1 as Anticancer DrugsCHEMMEDCHEM, Issue 12 2007Han Ang Dr. Abstract Ruthenium,arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione- S -transferase (GST) inhibitors with alternate modes of activity through the organometallic fragment, ultimately to provide targeted ruthenium-based anticancer drugs. Enzyme kinetics and electrospray mass spectrometry experiments using GST P1-1 and its cysteine-modified mutant forms revealed that the complexes are effective enzyme inhibitors, but they also rapidly inactivate the enzyme by covalent binding at Cys,47 and, to a lesser extent, Cys,101. They are highly effective against the GST Pi-positive A2780 and A2780cisR ovarian carcinoma cell lines, are among the most effective ruthenium complexes reported so far, and target ubiquitous GST Pi overexpressed in many cancers. [source] Surface modification of silicone intraocular lens by 2-methacryloyloxyethyl phosphoryl-choline binding to reduce Staphylococcus epidermidis adherenceCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 5 2007Xiao-Dan Huang MD Abstract Purpose:, To analyse the in vitro adherence of Staphylococcus epidermidis to the 2-methacryloyl oxyethyl phosphorylcholine (MPC)-modified silicone intraocular lens (IOL). Methods:, The test IOLs were modified by using an air plasma treatment to bind MPC to the surface. The control IOLs were not modified. Chemical changes on the IOL surface were analysed by X-ray photoelectron spectroscopy (XPS) to confirm the covalent binding of MPC. IOL hydrophilicity was determined by measuring the water contact angle. Two different techniques, direct counting of viable adherent bacteria released by sonication, and scanning electron microscopy (SEM), were used to observe and compare the adherence of S. epidermidis to the IOLs after 1- and 18-h incubation. Results:, XPS analysis confirmed that the test IOLs were surface-modified with MPC. The hydrophilicity of the IOLs was improved by surface modification, and the MPC-modified IOLs exhibited significantly reduced adhesion of S. epidermidis (P = 0.002) after an incubation period of 1 h. The SEM results showed that the MPC modification also suppressed the accumulation of bacteria and biofilm production after 18 h incubation. Conclusions:, MPC-modified hydrophilic silicone IOLs reduce bacterial adherence and colonization, and thus may help reduce the incidence of postoperative endophthalmitis. [source] | |||||||||||||||||||