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Elmar Heinzle

Publications and source records attributed to Elmar Heinzle.

12 recordsLinked to original sources
PubMed2003

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae.

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.

Amino Acids↗
PubMed2003

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation.

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.

Bioreactors↗
PubMed2003

PH-sensing 96-well microtitre plates for the characterization of acid production by dairy starter cultures.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37 degrees C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

Animals↗
PubMed2003

Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks.

On-line measurement of dissolved O2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O2-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum kLa values of 150 h(-1), from which maximum O2 transfer capacity of 33 mM h(-1) was calculated. Liquid volume and shaking frequency have a strong influence on kLa. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O2 supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O2 limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O2 sensing in shake-flasks, which are widely used in microbial cultivations.

Biosensing Techniques↗
PubMed2002

Identification and quantification of feathers, down, and hair of avian and mammalian origin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a fast method for the identification of the origin of native and chemically processed feathers, down, and hair and for distinguishing closely related species using enzyme digestion and MALDI-TOF mass spectrometry. Additionally we present two methods for the quantification of different identified bird and mammalian samples, respectively, in binary species mixtures. Without any prior cleaning or isolation of single proteins, enzymatical digests of feathers, down, and hair are performed. Fragments generated are analyzed by MALDI-TOF mass spectrometry, and peak groups of different selectivity are established for every animal species. For the identification of individual animal species, only unique species-specific peaks are accepted while for species classification of tinted or bleached hair, the group of semispecific peaks (SEMPs) is used. Samples from native animal species show a higher coincidence of digest peak masses of SEMPs than far-related species, indicating a phylogenetical relationship of the investigated structure proteins. Quantification of mixed binary avian samples is performed by identification of preweighed single feathers and down, followed by calculation of the gravimetric mass fractions. The composition of mixed binary mammalian samples is calculated from correlation of the quotient of the relative peak intensities or areas of these species and the quotient of corresponding gravimetric mass fractions. High accuracy is achieved by both quantification methods. The fast methods are well suited for industrial quality control for example.

Animals↗
PubMed2002

Correcting mass isotopomer distributions for naturally occurring isotopes.

In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.

Biomarkers↗
PubMed2002

Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix-assisted laser desorption/ionization mass spectrometry.

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.

Amino Acid Sequence↗
PubMed2002

Derivatization of small biomolecules for optimized matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.

Alcohols↗
PubMed2002

Methods for biocatalyst screening.

Biocatalysts are now widely accepted as useful alternative tools to classic organic synthetic techniques for the regio- and enantioselective synthesis under mild reaction conditions in many fields of chemistry. The development of techniques for the rational or evolutionary design of novel or modified enzymes has increased the need for fast and reliable methods for the identification of the most powerful catalysts. We present a short overview on screening techniques in this area. Beside classical methods such as spectrophotometry and fluorimetry, a number of new approaches like methods based on the measurement of pH changes or IR-thermography have been recently developed. Additionally the use of electrospray and matrix-assisted laser desorption/ionization mass spectrometry has gained increasing influence in this field of biotechnology.

Catalysis↗
PubMed2002

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.

Citric Acid Cycle↗
PubMedDate not supplied

Modeling of mixing in 96-well microplates observed with fluorescence indicators.

Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.

Acids↗