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Enzymatic Conversion (enzymatic + conversion)
Selected AbstractsModeling of Product Removal during Enzymatic Conversions by Using Affinity MoleculesBIOTECHNOLOGY PROGRESS, Issue 6 2007Daniël G. R. Halsema The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate. [source] Synthesis of deoxyadenosine-5,-(35S)-thiomonophosphate [dAMP(35S)] of high specific activity , a key intermediate for the synthesis of Sp-deoxyadenosine-5,-(alpha; - 35S)-thiotriphosphate [Sp-dATP (, - 35S)]JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 1 2002K. M. Mathew Abstract Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid and stoichiometric proportions of N, N,-di- p -tolylcarbodiimide in anhydrous pyridine to give deoxyadenosine-5,-monophosphite 2. The latter was activated with trimethylsilyl chloride followed by sulphurisation with elemental 35S (specific activity>1000 Ci/mmol) in toluene solution to give deoxyadenosine-5,-(35S)-thiomonophosphate [dAMP(35S)] 3. Enzymatic conversion of deoxyadenosine-5,-(35S)-thiomonophosphate to Sp-deoxyadenosine-5,-(, - 35S)-thiotriphosphate [Sp-dATP (, - 35S)] 5 was carried out following a standard reaction protocol. Copyright © 2001 John Wiley & Sons, Ltd. [source] Kinetic analysis of hyaluronidase activity using a bioactive MRI contrast agentCONTRAST MEDIA & MOLECULAR IMAGING, Issue 3 2006Liora Shiftan Abstract One of the attractions of molecular imaging using ,smart' bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R2 as a result of hyaluronidase activity. The changes in R2 and the measured relaxivity of intact HA-GdDTPA-beads (r2B) and HA-GdDTPA fragments (r2D) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 °C with peak activity at 25 °C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases. Copyright © 2006 John Wiley & Sons, Ltd. [source] Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3FEBS JOURNAL, Issue 18 2009Xiao Luo Insulin-like peptide 3 (INSL3), which is primarily expressed in the Leydig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3, we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body, purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide. [source] Synthesis of [13C]-isotopomers of indole and tryptophan for use in the analysis of indole-3-acetic acid biosynthesisJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 10 2004Neboj, a Ili Abstract The direct conversion of indole to indole-3-acetic acid without tryptophan as an intermediate has previously been shown to occur in vivo, as well as in vitro, with seedlings of plants. In order to facilitate the purification of the enzymes that carry out the enzymatic synthesis of indole-3-acetic acid from labeled indole, it was necessary to develop an assay that had both high sensitivity and analytical precision. To obtain the required analytical resolution and to allow definitive product identification, [13C6]indole was synthesized for use in GC-MS assays of the enzymatic conversion. Plants have been shown to be able to synthesize indole-3-acetic acid either directly from indole as well as by degradation of tryptophan. Thus, in order to allow the biochemical discrimination between these processes, the synthesized [13C6]indole was used as a starting material for a novel enzymatic synthesis of [13C]isotopomers of L -tryptophan labeled at specific positions. Together, these isotope labeled indolic compounds offer powerful new approaches to understanding and differentiating routes of indole-3-acetic acid biosynthesis in vitro and in vivo. Copyright © 2004 John Wiley & Sons, Ltd. [source] A novel transverse push,pull microprobe: in vitro characterization and in vivo demonstration of the enzymatic production of adenosine in the spinal cord dorsal hornJOURNAL OF NEUROCHEMISTRY, Issue 1 2001Shawnna L. Patterson Adenosine produces analgesia in the spinal cord and can be formed extracellularly through enzymatic conversion of adenine nucleotides. A transverse push,pull microprobe was developed and characterized to sample extracellular adenosine concentrations of the dorsal horn of the rat spinal cord. Samples collected via this sampling technique reveal that AMP is converted to adenosine in the dorsal horn. This conversion is decreased by the ecto-5,-nucleotidase inhibitor, ,,,-methylene ADP. Related behavioral studies demonstrate that AMP administered directly to the spinal cord can reverse the secondary mechanical hyperalgesia characteristic of the intradermal capsaicin model of inflammatory pain. The specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) inhibits the antihyperalgesia produced by AMP. This research introduces a novel microprobe that can be used as an adjunct sampling technique to microdialysis and push,pull cannulas. Furthermore, we conclude that AMP is converted to adenosine in the dorsal horn of the spinal cord by ecto-5,-nucleotidase and subsequently may be one source of adenosine, acting through adenosine A1 receptors in the dorsal horn of the spinal cord, which produce antihyperalgesia. [source] Comparison of suppressive potency between prednisolone and prednisolone sodium succinate against mitogen-induced blastogenesis of human peripheral blood mononuclear cells in-vitroJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2001Kentaro Sugiyama Clinically, both prednisolone and prednisolone sodium succinate are widely used as immunosuppressive agents for the treatment of various allergic disorders. However, whether prednisolone sodium succinate itself has immunosuppressive or anti-inflammatory effects is unclear, and prednisolone sodium succinate may exhibit its efficacy only after hydrolytic conversion to prednisolone in-vivo. If this is the case, the impairment of prednisolone sodium succinate conversion to prednisolone in some clinical conditions may attenuate the efficacy of prednisolone sodium succinate. We therefore compared the pharmacological efficacy of prednisolone with that of prednisolone sodium succinate in-vitro using human peripheral blood mononuclear cells (PBMCs). PBMCs were obtained from 5 healthy subjects and 1 patient with pneumonia. The cells were incubated in the presence of concanavalin A and the cell growth was estimated by 3-(4,5-dimethyl thiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Both prednisolone and prednisolone sodium succinate dose-dependently suppressed PBMC blastogenesis. Mean (s.d.) prednisolone and prednisolone sodium succinate IC50 (concentration of drug that gave 50% inhibition of cell growth) values were 580.0 (1037.9) and 3237.1 (4627.3) nm, respectively. The ratio of prednisolone IC50/prednisolone sodium succinate IC50 ranged from 0.005 to 0.230. Thus, prednisolone sodium succinate potency was markedly lower than that of prednisolone. After incubation of PBMCs with 100 ,m prednisolone sodium succinate, 22.7,42.9 ,m prednisolone was liberated into the culture medium, as determined by HPLC. The ratio of prednisolone liberation from prednisolone sodium succinate was not affected by the presence of fetal bovine serum or PBMC, or both, in the culture medium. These results suggested that the PBMC-suppressive effects of prednisolone sodium succinate might be due, at least partially, to prednisolone liberated from prednisolone sodium succinate into the culture medium. Prednisolone sodium succinate can be converted to prednisolone in the absence of serum or PBMCs, but the ratio of this conversion was very slow (t£frac12; > 4 days). Therefore, impairment of the enzymatic conversion of prednisolone sodium succinate to prednisolone in some pathological conditions such as liver diseases may result in attenuation of the clinical efficacy of prednisolone sodium succinate. [source] An improved high-performance liquid chromatography method for quantification of methotrexate polyglutamates in red blood cells of children with juvenile idiopathic arthritisBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 3 2009Hroch Abstract Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463,nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with ,-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6,ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05,m, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25,400,nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9,nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis. Copyright © 2009 John Wiley & Sons, Ltd. [source] Understanding the key factors for enzymatic conversion of pretreated lignocellulose by partial least square analysisBIOTECHNOLOGY PROGRESS, Issue 2 2010Renliang Huang Abstract The relationship between the physicochemical properties of lignocellulosic substrates and enzyme digestion is still not well known. After different pretreatments, cellulase hydrolysis and measurements of physicochemical characteristics by column solute exclusion, particle size analysis, X-ray diffraction, Fourier transform infrared spectroscopy and solid state 13C nuclear magnetic resonance were performed in this study. Partial least squares was then applied to seek the key factors limiting the rate and extent of cellulose digestion. According to the PLS results, the most important factor for cellulose digestion was accessible interior surface area, followed by delignification and the destruction of the hydrogen bonds. The cellulose digestion at 2 and 24 hr were improved with the increased accessibility of interior surface area to the reporter molecules of 5.1-nm diameter. Removal of lignin and breaking of hydrogen bonds were also found to significantly promote cellulose conversion. Other properties, including the breakdown of intramolecular hydrogen bonds, cellulose crystallinity, and hemicellulose content, had less effect on the efficiency of enzymatic hydrolysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Enzymatic digestion of liquid hot water pretreated hybrid poplarBIOTECHNOLOGY PROGRESS, Issue 2 2009Youngmi Kim Abstract Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot-washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot-washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar-to-ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Modeling of Product Removal during Enzymatic Conversions by Using Affinity MoleculesBIOTECHNOLOGY PROGRESS, Issue 6 2007Daniël G. R. Halsema The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate. [source] Chirality sensing with synthetic pores,CHIRALITY, Issue 3-4 2008Hiroyuki Tanaka Abstract A concept to determine enantiomeric excess with synthetic multifunctional pores is introduced. To do so, the poor stereoselectivity of molecular recognition by stimuli-responsive pores is coupled with the stereospecificity of enzymes. With substrates as good and products as poor pore blockers, enzymatic conversion of one enantiomer is shown to readily reveal the concentration of the other one. Calculations suggest that high substrate/product discrimination by the synthetic pores may provide access to the accurate detection of the extreme enantiomeric excess that is of interest in chemistry, pharmacology, and medicine, but otherwise possibly problematic to detect. Validity of the introduced concept is experimentally confirmed with poly- L -glutamate and poly- D -glutamate as enantiomeric substrates with high blockage efficiency, L -glutamate and D -glutamate as enantiomeric products with poor blockage efficiency, subtilisin A as enzyme, and a classical rigid-rod ,-barrel as synthetic pore. Chirality, 2008. © 2007 Wiley-Liss, Inc. [source] Recent advances in capillary electrophoresis and capillary electrochromatography of peptidesELECTROPHORESIS, Issue 22-23 2003Václav Ka Abstract An overview of the recent developments in the applications of high-performance capillary electromigration methods, namely zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides is presented. New approaches to the theoretical description and experimental verification of the electromigration behavior of peptides and the methodological aspects of capillary electroseparations of peptides, such as rational selection of separation conditions, sample treatment, and suppression of adsorption, are discussed, and new developments in individual separation modes and new designs of detection systems applied to peptide separations are shown. Several types of applications of capillary electromigration methods to peptide analysis are presented: quality control and purity tests, determination in biomatrices, monitoring of physical and chemical changes and enzymatic conversions, amino acid and sequence analysis, and peptide mapping. The examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated. [source] Modeling of Product Removal during Enzymatic Conversions by Using Affinity MoleculesBIOTECHNOLOGY PROGRESS, Issue 6 2007Daniël G. R. Halsema The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate. [source] | ||||||||||||||||