A Consensus Match Scoring System thatis Correlated with Biological Functionality

The C;-scoring system is suitable for the identification of putative functional transcription factor binding sites solely by sequence analysis. This is a very important feature since it allows preselection of candidate bindingsites for experimental analysis from uncharacterized genomic sequences. Data derived from the analysis of almost 2.4 million nucleotides of genomic sequences show a good correlation of C;-scoring with known biological function. High-scoring bindingsites are clearly over-represented in putative control regions of the genomic sequences. Known functional bindingsites cluster in the same regions. Furthermore, we demonstrate high C;-scores to correlate with biological functionality of 26 individual binding sites for three completely unrelated transcription factors. Consensus matches knownto be either nonfunctional or to bind their correspondingprotein factors with highly reduced affinity are low-scorers in ourrating

PROBING THE CARBOHYDRATE SIDE CHAINS OF RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR

The glycosylation of recombinant tissue plasminogen activator derived from transfected CHO cells was assessed using glycosyltransferases to probe for terminal Gal and GlcNAc residues and solid-state lectin binding assays. Analysis of the oligosaccharides released from each glycosylation site by hydrazinolysis by Con A affinity chromatography/ anion exchange HPLC/ BioGel P4 gel filtration corroborated the glycosyltransferase and lectin-binding assay data. Significant undersialylation of the oligosaccharides of t-PA and less than 1% of free terminal GlcNAc residues was indicated. The above assays could be useful in monitoring glycosylation status during quality control in recombinant glycoprotein production

RAPID DETERMINATION OF BIOMASSACTIVITY BY FLOW INJECTION ANALYSIS

A flow injection analysis (FIA) method using the metabolic reduction of a redox mediator by microorganisms and subsequent electrochemical detection of the reduced mediator was applied to high cell density fermentations of E. coli. Good correlation to the oxygen uptake rate (OUR) was found and thus the FIA methodwas suitable to monitor the biomassactivity on-line

CLONING, SEQUENCING AND REGULATION OF THE LIPASE GENE FROM PSEUDOMONASSP. M-12-33

Thelipase gene from Pseudomonas sp. M-12-33 was cloned. The cloned DNA was 2.9 Kb in length, which was essential for production of the lipase and contained two open reading frames, lipA and lipX. The lipA gene was supposed to comprise 1092 nucleotides and give a preproprotein of 364 aminoacids which was then processed to a mature lipase protein of 320 amino acids. On the other hand, the lipX gene was assumedto code a protein of 344 amino acids concerned with someregulation of the enzyme production

Development of Microbial Sensors for Determination of Xenobiotics

Amperometric biosensors using immobilised microbial cells as the biological component were developed for the determination of biphenyl and its chlorinated derivatives. Measurements were based on the respiratory activity of the microbial cells. The influence of different organic solvents on the respiratory activity was analysed. Different membranes were used for immobilising the cells across the face of an oxygen electrode; to determine whetherthe polarity of the membrane had an influence on the substrate degraded

APPLICATION OF NADH OXIDASEIN FIBRE OPTIC BIOSENSORS

A newfully reversible fibre-optic detection system based on the detection of NADH-fluorescence is presented. NADH oxidase (EC: 1.6.99.3) was used to regenerate NADH thatis needed for the oxidizing reaction of alcohols and aldehyds by different dehydrogenases.In the oxidation reaction NAD* wasreduced to NADH and the increase of fluorescence was monitored bya fibre-optic detection system. The NADH-fluorescence decreased in the absence of substrate due to the oxidation of NADH by NADH oxidase. Different types of NADH oxidase (Thermus thermophilus, Thermus aquaticus und Bacillus licheniformis) were studied in respectto their application in optical sensors. Only NADH oxidaseof B. licheniformis proved to beactive and stable at any assay conditions even in the absenceof FAD

LIPOLYTIC ENZYMES SEPARATION AND PURIFICATION THROUGH FUNCTIONALIZED SYNTHETIC POLYMERS

A new class of functionalized synthetic polymers was prepared for the purification of lipases by a single step affinity chromatography. Polyvinyl alcohol polymers were crosslinked with epichlorohydrin and esterified with fatty acids of different length. The resulting resins were characterized by infrared spectroscopy, electron microscopy and titration of the hydrolysed product to evaluate the degree of esterification. The attention was focused on lauryl ester of polyvinyl alcohol as commercial preparation of Candida cylindracea lipase showed the highest enzyme affinity for esters of fatty acids with a linear chain ranging from 8 to 12 carbon atoms. Chromatographic lipase purification trials were performed on a 7 cm x 1.6 cm i.d. column. Good separation conditions were found by utilizing stepwise increase in CHAPS (3-(3-(cholamido-propyl)-dimetil-ammonio)-1-propan sulfonate) concentration in HEPS/EDTA buffer. The lipase obtained by elution with CHAPS 6.0 mmol/L showed a high purity, as established by SDS-PAGE, where an unique band with a molecular weight of 60.000 was identified

PROTEIN O-GLYCOSYLATION AND SEXUAL AGGLUTININS IN THE YEAST S.CEREVISIAE

The features of protein O-glycosylation in yeast are summarized. - The a and a-agglutinin of haploid S.cerevisiae cells have been purified and their one to one interaction has been studied in vitro; their carbohydrate moieties do not seem to be essential. Both the corresponding genes have been cloned and sequenced

THE USE OF BIOSENSORS IN DETERMINATION OF DIFFERENT SUBSTANCES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A rapid, simple and economic method was developed, which combines the specificity of enzymes with the high sensitivity of HPLC. Therefore sample pretreatment is reduced to simple dilution or extraction steps. In this work oxidases for alcohol, glucose, oxalic acid, ascorbic acid and galactose were immobilized and used as biosensors. Food samples were fruit juices and vegetable products, soft drinks and wine. The detection limit e.g. for oxalic acid was in the picomol range

ENZYMATIC MULTI-CHANNEL-FIA METHODS FOR ONLINE FERMENTATION MONITORING AND CONTROL

A multichannel FIA system with high reliability and flexibility was developed for on-line process monitoring and control of fed batch and continuous fermentations. Glucose, L-Lactate, D-a and L-a-Hydroxybutyric acid(D- und LHBS), Isoleucin, Leucin, Ethanol, Acetaldehyde and Ammonia are enzymatically determined with fluorimetric detection. For all assays dehydrogenases alone or coupled with a second enzyme are immobilized in packed bed tube reactors. In the proposed parallel configuration of enzyme reactors only one detector is necessary. In a miniaturized and modular on-line sampling system the sample solution is diluted, conditioned and purified from interfering substances. The proposed FIA set up allows the adaptation of the different analyte concentrations to the ranges of the corresponding enzymatic assays. A freely programmable valve configuration guarantees a high flexibility in solving different problems of process analysis. The whole FIA-equipmentis controlled by an user friendly software running on a personal computer.The process analytical regime consists of on-line analysis, calibration- and recalibration cycles. The control of these cycles, the detctor signal evaluation and the calculation of the analytical results are implemented fully automatic