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  • 1
    Electronic Resource
    Electronic Resource
    Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 111-129 
    Details
    ISSN: 0006-3592
    Keywords: intracellular fluxes ; metabolite balance ; carbon labeling balance ; lysine ; anaplerotic reactions ; NMR spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of 1H-detected 13C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-13C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. © 1996 John Wiley & Sons, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 118-135 
    Details
    ISSN: 0006-3592
    Keywords: stationary flux estimation ; sensitivity analysis ; covariance analysis ; nonlinear statistics ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic carbon labelling experiments enable a large amount of extracellular fluxes and intracellular carbon isotope enrichments to be measured. Since the relation between the measured quantities and the unknown intracellular metabolic fluxes is given by bilinear balance equations, flux determination from this data set requires the numerical solution of a nonlinear inverse problem. To this end, a general algorithm for flux estimation from metabolic carbon labelling experiments based on the least squares approach is developed in this contribution and complemented by appropriate tools for statistical analysis. The linearization technique usually applied for the computation of nonlinear confidence regions is shown to be inappropriate in the case of large exchange fluxes. For this reason a sophisticated compactification transformation technique for nonlinear statistical analysis is developed. Statistical analysis is then performed by computing appropriate statistical quality measures like output sensitivities, parameter sensitivities and the parameter covariance matrix. This allows one to determine the order of magnitude of exchange fluxes in most practical situations. An application study with a large data set from lysine-producing Corynebacterium glutamicum demonstrates the power and limitations of the carbon-labelling technique. It is shown that all intracellular fluxes in central metabolism can be quantitated without assumptions on intracellular energy yields. At the same time several exchange fluxes are determined which is invaluable information for metabolic engineering. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 118-135, 1997.
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  • 3
    Electronic Resource
    Electronic Resource
    Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 624-635 
    Details
    ISSN: 0006-3592
    Keywords: NMR spectroscopy ; membrane cyclone reactor ; oxygen transfer ; Zymomonas mobilis ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients kLa of more than 1.0 s-1 are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate 〉15,000 mg O2 ·(L h)-1] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo 31P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo 13C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 gcell dry weight · L-1, the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-13C]-glucose pulse.
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  • 4
    Electronic Resource
    Electronic Resource
    Response of the central metabolism of Corynebacterium glutamicum to different flux burdens (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 168-180 
    Details
    ISSN: 0006-3592
    Keywords: metabolic fluxes ; metabolite balance ; NMR spectroscopy ; amino acid production ; bidirectional fluxes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-13C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of 13C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate ↔ sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate ↔ fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.
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  • 5
    Electronic Resource
    Electronic Resource
    Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 101-117 
    Details
    ISSN: 0006-3592
    Keywords: stationary flux analysis ; metabolite flux balancing ; 13C isotope labeling experiments ; NMR ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The extension of metabolite balancing with carbon labeling experiments, as described by Marx et al. (Biotechnol. Bioeng. 49: 11-29), results in a much more detailed stationary metabolic flux analysis. As opposed to basic metabolite flux balancing alone, this method enables both flux directions of bidirectional reaction steps to be quantitated. However, the mathematical treatment of carbon labeling systems is much more complicated, because it requires the solution of numerous balance equations that are bilinear with respect to fluxes and fractional labeling. In this study, a universal modeling framework is presented for describing the metabolite and carbon atom flux in a metabolic network. Bidirectional reaction steps are extensively treated and their impact on the system's labeling state is investigated. Various kinds of modeling assumptions, as usually made for metabolic fluxes, are expressed by linear constraint equations. A numerical algorithm for the solution of the resulting linear constrained set of nonlinear equations is developed. The numerical stability problems caused by large bidirectional fluxes are solved by a specially developed transformation method. Finally, the simulation of carbon labeling experiments is facilitated by a flexible software tool for network synthesis. An illustrative simulation study on flux identifiability from available flux and labeling measurements in the cyclic pentose phosphate pathway of a recombinant strain of Zymomonas mobilis concludes this contribution. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 101-117, 1997.
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  • 6
    Electronic Resource
    Electronic Resource
    13C tracer experiments and metabolite balancing for metabolic flux analysis: Comparing two approaches (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 254-257 
    Details
    ISSN: 0006-3592
    Keywords: metabolic flux analysis ; 13C tracer experiments ; fractional enrichment ; NADH ; NADPH ; pentose phosphate pathway ; Aspergillus oryzae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for intracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends heavily on the correctness of the assumed stoichiometry including the presence of all reactions with a noticeable impact on the model metabolite balances. Determination of fluxes in complex metabolic networks often requires the inclusion of NADH and NADPH balances, which are subject to controversial debate. Transhydrogenation reactions that transfer reduction equivalents from NADH to NADPH or vice versa can usually not be included in the stoichiometric model, because they result in singularities in the stoichiometric matrix. However, it is the NADPH balance that, to a large extent, determines the calculated flux through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using 13C tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well established stoichiometric equations and measurements of the labeling state of intracellular metabolites. Neither NADH/NADPH balancing nor assumptions on energy yields need to be included to determine the intracellular fluxes. Because metabolite balancing methods and the use of 13C labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviations in the results. Flux analysis based entirely on metabolite balancing and flux analysis, including labeling information, have been performed independently for a wild-type strain of Aspergillus oryzae producing α-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on the intracellular flux distribution. The two different approaches to the calculation of fluxes are compared and deviations in the results are discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:254-257, 1998.
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  • 7
    Electronic Resource
    Electronic Resource
    Rearrangements in the electron-impact induced fragmentation of some β-aroyl-α-methylpropionic acids (1971)
    Chichester : Wiley-Blackwell
    Biological Mass Spectrometry 5 (1971), S. 1081-1084 
    Details
    ISSN: 0030-493X
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A complex rearrangement on electron-impact for β-aroyl-α-methylpropionic acids, involving both hydrogen and hydroxy migration followed by loss of carbon monoxide and allyl radical, is described and discussed. The rearrangement process, resulting in an ion [ArCOOH2]+, is favoured by electron-withdrawing substituents in the aromatic ring.
    Additional Material: 2 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/oms.1210050907
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  • 8
    Electronic Resource
    Electronic Resource
    Studies on the mechanism of electron impact induced SH radical loss from the diastereoisomeric 2-dimethylamino-2-thiono-4-methyl-1,3,2-dioxaphosphorinanes (1980)
    Chichester : Wiley-Blackwell
    Biological Mass Spectrometry 15 (1980), S. 105-108 
    Details
    ISSN: 0030-493X
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The intensity differences between the [M—HS]+ peaks in the mass spectra of diastereoisomeric 2-dimethylamino-2-thiono-4-methyl-1,3,2-dioxaphosphorinanes have been rationalized in terms of fragmentation mechanisms and ion structures. Metastable characteristics together with D labelling experiments support these rationalizations.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/oms.1210150210
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  • 9
    Electronic Resource
    Electronic Resource
    Carbon-13 NMR study of enamines and enamino ketones. A direct observation of the conformation of the 2-methylcyclohexanone enamine tautomers (1975)
    Chichester : Wiley-Blackwell
    Organic Magnetic Resonance 7 (1975), S. 433-441 
    Details
    ISSN: 0030-4921
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The carbon-13 spectra of a series of cyclohexanone, cyclopentanone and 2-methylcyclohexanone enamines are reported. The influence of the amino component was studied in a series of enamino ketones. The spectra reflect the amount of p-π overlap in the molecules and their conformation. Differences between 5-and 6-membered carbonyl components are evaluated.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrc.1270070908
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  • 10
    Electronic Resource
    Electronic Resource
    Influence of the bulk and surface morphology on adhesion of polystyrene-inter-poly-cross-2-ethylhexyl-methacrylate films and particles (1996)
    Bognor Regis [u.a.] : Wiley-Blackwell
    Journal of Polymer Science Part B: Polymer Physics 34 (1996), S. 1839-1852 
    Details
    ISSN: 0887-6266
    Keywords: adhesion ; interpenetrating polymer networks (semi-) ; polystyrene ; polymethacrylate ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Physics
    Notes: The adhesion behavior of semi-interpenetrating polymer networks (semi-IPNs) of linear polystyrene (PS) in crosslinked poly-2-ethylhexylmethacrylate (EHMA) was studied by variation of the bulk and surface morphology, i.e., domain size, continuity, and concentration in the domains. Semi-IPNs were prepared by liquid-liquid demixing upon cooling of a homogeneous solution of PS in methacrylate monomer, followed by gelation of the PS-rich phase and UV polymerization of the methacrylate resin. Welding of films allowed the preparation of larger objects provided that (1) the samples were phase separated to a high degree and contained domains with a high PS concentration (〉90%) and (2) polystyrene was present at the interface. For semi-IPN films, a linear dependence of the adhesion strength on the (crack healing time)1/4 was obtained. Based on these considerations, a process was developed to obtain melt-processable semi-IPN particles, by quenching droplets of the polymer solution into a cold liquid. These particles obtained a PS-rich skin layer and showed good adhesion after blending with a thermoplast. © 1996 John Wiley & Sons, Inc.
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