Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH).

Background: A new method was established to characterize the binding kinetics of DNA toward layered double hydroxides (LDHs). The setup consisted of a newly developed sampling tube that allows the injection of analyte during the flow cytometric measurement.

Methods: Layered double hydroxides consist of cationic metal hydroxide layers and exchangeable interlayer anions.

Visualizing transport processes at liquid-liquid interfaces--the application of laser-induced fluorescence.

Modeling of liquid-liquid extraction processes involves the concentration of the extracted component directly at the interface. Currently, only very few and specialized methods are available for the direct measurement of these concentrations. Therefore a new, fluorescence-based measurement system with a high spatial resolution and a broad application spectrum was developed and tested.

Chemometric modelling with two-dimensional fluorescence data for Claviceps purpurea bioprocess characterization.

Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy and the application of chemometric methods accomplish these goals. In order to demonstrate how time-consuming off-line analysis methods can be replaced for bioprocess monitoring, fluorescence measurements were performed during different cultivations of the fungus Claviceps purpurea.

Multi parameter in vitro testing of ratjadone using flow cytometry.

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action.

The total synthesis of (-)-callystatin A.

Callystatin A is a prominent member of a class of natural products which display promising growth inhibition of cancer cells in their biological profile. The challenging structure and the interesting biological activity of (-)-callystatin A fueled our interest in the synthesis of this marine natural product. We achieved the total synthesis using a highly convergent approach joining four subunits together with a Wittig olefination, a selective Heck reaction and an aldol reaction as the pivotal steps.

Chemometric modelling based on 2D-fluorescence spectra without a calibration measurement.

Motivation: 2D fluorescence spectra provide information from intracellular compounds. Fluorophores like trytophan, tyrosine and phenylalanin as well as NADH and flavins make the corresponding measurement systems very important for bioprocess supervision and control. The evaluation is usually based on chemometric modelling using for their calibration procedure off-line measurements of the desired process variables.

Controlled enzymatic removal of damaging casein layers on medieval wall paintings.

A new, gentle enzymatic method was developed for a controlled removal of casein layers from medieval wall paintings. These casein layers were applied over the last 60 years on wall paintings in order to decrease substantial damage due to a peeling off of the frescoes from the roughcast surface due to environmental effects. However, due to the aging of the casein layers (at 40-50 years), a more drastic peeling occurred and the danger of total destruction of the wall paintings is severe.

In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring.

Optical sensors appear to be very promising for different applications in modern biotechnology. They offer the possibility to interface all the well known optical analysis techniques to bioprocesses via fiber optical cables. Thus, high sophisticated and sensitive optical analysis techniques can be coupled to a bioprocess via these light signal transporting fibers.

Basic examinations on chemical pre-oxidation by ozone for enhancing bioremediation of phenanthrene contaminated soils.

Biological treatment of polycyclic aromatic hydrocarbons (PAH) has been demonstrated to be a feasible and common remediation technology which has been successfully applied to the clean-up of contaminated soils. Because bioavailability of the contaminants is of great importance for a successful bioremediation, a chemical pre-oxidation step by ozone was tested to enhance the subsequent biodegradation steps. Oxidation of PAH by ozone should result in reaction products that have a better solubility in water and thus a better bioavailability.

Flow cytometry in biotechnology.

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium.

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine.

The enantioselective hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide using lipases.

A new experimental high-pressure-unit was constructed for the enantioselective enzymatic hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester (a precursor for biological interesting substances) in a biphasic buffer/SCCO(2)-system. One objective is to take advantage of the solubility differences of the substrate and the produced acid. Thus the different solubilities of the substrates and the products in the different phases were studied regarding to an overall process integration.

Oxygen monitoring in supercritical carbon dioxide using a fibre optic sensor.

Investigations of enzymatic reactions in supercritical CO(2) are often hindered by the high pressure involved in these processes, making reaction monitoring extremely difficult. This paper describes the implementation of a fiber optic based oxygen sensor into a high pressure reactor for supercritical carbon dioxide. The sensor is pressure resistant, working in supercritical carbon dioxide and reusable after depressurisation.

Microbial degradation of phenanthrene by addition of a sophorolipid mixture.

The influence of sophorolipids on microbial degradation of poorly soluble phenanthrene in liquid and soil suspension culture was evaluated in the work presented. Experiments were carried out in two parts. In the first part, important basic physico-chemical characteristics of the biosurfactant and the pollutant used were determined.

Monitoring and control of industrial downstream processing of sugar beet molasses.

In present work the determination of several amino acids during the industrial chromatographic desugarisation of molasses is presented. The use of innovative biosensor systems for highly specific detection of serine is described. Using two-dimensional fluorescence spectrometry, a non-invasive method for the determination of several product fractions could be established in an industrial chromatographic procedure.

Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes.

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.

Fluorescence monitoring during cultivation of Enterobacter aerogenes at different oxygen levels.

On-line monitoring of NAD(P)H fluorescence and 2D fluorescence spectroscopy was performed with Enterobacter aerogenes, a bacterium sensitive to oxygen availability. The organism was grown in a reactor under low and high dissolved oxygen concentrations and circulated through a bypass attached to the reactor. Under low dissolved oxygen concentration in the reactor, NAD(P)H fluorescence in the reactor and the bypass showed a deviation, but not when the dissolved oxygen level in the reactor was high.

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively.

A simple method for the isolation and purification of L-asparaginase from Enterobacter aerogenes.

L-Asparaginase from Enterobacter aerogenes was purified by a simple method involving sonication of the crude cell mass, gel filtration with Sephacryl S-100 as the separating material, followed by ultrafiltration. Recent methods involve complex purification procedures of 5-6 steps. The isolation process resulted in 10-fold purification of the enzyme with a specific activity of 55 IU/mg protein and recovery of 54%.

A model of molecular circadian clocks: multiple mechanisms for phase shifting and a requirement for strong nonlinear interactions.

A fundamental question in the field of circadian rhythms concerns the biochemical and molecular nature of the oscillator. There is strong evidence that circadian oscillators are cell autonomous and rely on periodic gene expression. In Drosophila, Neurospora, Aplysia, and vertebrates, circadian oscillators are thought to be based on molecular autoregulatory loops composed of transcription, translation, and negative feedback by proteins on nuclear transcription.

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining.

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acid were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry.

In situ microscopy for on-line determination of biomass.

A sensor is presented, which allows on-line microscopic observation of microorganisms during fermentations in bioreactors. This sensor, an In Situ Microscope (ISM) consists of a direct-light microscope with a measuring chamber, integrated in a 25 mm stainless steel tube, two CCD-cameras, and two frame-grabbers. The data obtained are processed by an automatic image analysis system.

Thermal biosensors in biotechnology.

The application of enzyme thermistor devices for the continuous monitoring of enzymatic processes is described. Different hardware concepts are presented and discussed, practical results are also given. These devices were used to analyze the enantiomeric excess in biotransformation processes and for thermal immunoanalysis.

Flow injection analysis system for the supervision of industrial chromatographic downstream processing in biotechnology.

Sugar beet molasses is a natural resource for various products used in daily life, ranging from sucrose to amino acids for pharmaceutical industry. The separation of molasses into these high value components is performed on a large scale by ion exchange/exclusion chromatography. A biosensor system was set up for the "in time" analysis of serine and sucrose during molasses desugarisation.