Now showing items 21-40 of 4404


      Schulmeister, Thomas; Schubert, Florian; Central Institute of Molecular Biology, Academy of Sciences of the GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      The advantages of computer assisted enzyme electrode design are demonstrated for the case of electrodes with cycling reactions. 1. Introduction The number of really existing amperometric enzyme electrodes is increasing. The arrangements become more and more complicated: - several enzymes are combined to enzyme systems — non-stationary operational modes are used — enzymes with non-linear kinetics are applied The designer of new enzyme electrodes can be much supported by making available suitable mathematical models: - calculation of all concentration profiles involved in the enzyme membrane (understanding of the basic principles) — prediction of the dynamic behaviour of the sensor, e.g. current/time behaviour, response time, linear measuring range. - optimizing of the sensor design (cost saving use of enzymes, achievement of given device parameters, use of commercial enzymes). This aim can be realized by a user-friendly software package which operates in the laboratory on a personal computer. The corresponding mathematical fundamentals are available: — one-dimensional reaction/diffusion systems are proved to be good - linear models have been investigated extensively, numerous explicit formulae have been derived — numerical methods and the tools for software development (compiler, libraries, graphics) are international standard.

      Hintsche, Rainer; Scheller, Frieder; Central Institute of Molecular Biology , Aacdemy of Sciences of the GDR, Robert—Roessle-Strasse 10. 1115 Berlin — Buch, GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Some methods to cover electrochemical sensors based on silicon technology with enzyme membranes using different polymer systems are known. The main requirements of enzyme membranes at electrochemical sensor elements based on silicon technology, such as ion sensitive field effect transistors (ISFETs) and thin film noble metal electrodes (TFMEs), are good adhesion at the sensor surface and minimal diffusion resistance for substrates and products. Furthermore the amount of enzyme molecules immobilized at the active surface of the sensor element influences the functional Parameters of the biosensor, e.g. lifetime, sensitivity, linearity, and response time, which should be in the same range as for the enzyme membranes of first generation biosensors. In this paper we describe new covering procedures that have been studied in order to find out technologies compatible to microelectronic Production technology used for the fabrication of TFMEs (FIG 1) and multigate ISFETs.
    • An in situ Fermenter Probe for Bakers Y east Propagation Monitoring

      Bradley, Joanne; Turner, Anthony P. F.; Schmid, Rolf D.; GBF - Gesellschaft fiir Biotechnologische Forschung, Abteilung Enzymtechnologie, Mascheroder Weg 1, D-3300 Braunschweig, (FRG). * Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire, MK43 OAL, (UK) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      A fermenter probe was developed based on the immobilisation of glucose oxidase at an amperometric mediated graphite electrode. The response characteristics of the probe made it readily applicable to continuous monitoring of glucose concentration during bakers yeast propagation.

      Wollenberger, Ulla; Scheller, Frieder; Pawlowa, Mariana; Müller, Hans-Georg; Risinger, Lars; Gorton, Lo; Central Institute of Molecular Biology, Academy of Sciences of the GDR, 1115 Berlin-Buch, Analytical Chemistry, University of Lund, 5-22100 Lund, Sweden (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      The determination of L-glutamate is important in fermentation control in foodstuff industry, because many kinds af food contain Glutamate as an essential flaveur compound, Furthermore, determination of Lglutamine demanded in on-line control cof mammalian cell culture, Glutamate produced in enzyme reactions, @.g., by a transaminas ’, can be & Measure of the respective enzyme activity. These data are of high value in the diagnosis of heart and liver deseases.

      Berg, Peter; Näbauer, Anton; Ruge, Ingolf; Fraunhofer-Institut für Festkörpertechnologie, Paul-Gerhardt-Allee 42, 8000 München 60, West Germany * Lehrstuhl für Integrierte Schaltungen, Technische Universität München, Arcisstr. 21, 8000 München 2, West Germany (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      As on the one handa field effect transistor (FET) is a widespreadelectronic device for a potentiometric measuring of electrical phenomena, on the other hand change of dipole moments or accumulation of net charge occur as a consequence of a biological reaction, the combination of an ion sensitive field effect transistor (ISFET) with a biological layer, the so-called Bio-FET, is an excellent device for measuring concentrations of biological substances in a sample solution. The ISFET,fabricated by the semiconductor technology with low fabrication costs, offers the possibility of integrating signal processing together with the FET-sensor. Principle of function, layout of the sensor chip, integrated multi sensor array, experimental results are the topics treatedin this paper.

      Brückel, J.; Zier, H.; Kerner, W.; Pfeiffer, E. F.; Department of Internal Medicine I, University of Ulm, F.R.Germany (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      As we have described previously, an amperometric enzyme electrode was developed, based on the detection of hydrogen peroxide generated by the oxidation of glucose (Kerner et al., Horm.Metab.Res.Suppl.20: 8-13,1988). This needle-type electrode was implanted into sc.tissue of sheep. It is shown that the sensor current is closely related to the course of blood glucose. For measurement of glucose in blood comparable to the Biostator, an electrode was further modified and integrated into a flow-chamber system. In vitro experiments demonstrated its qualification. Monitoring of blood glucose was performed over 24 hours.

      Trott-Kriegeskorte, Gundula; Renneberg, Reinhard; Pawlowa, Mariana; Schubert, Florian; Hammer, Joachim; Jäger, Volker; Wagner, Roland; Schmid, Rolf D.; Scheller, Frieder W.; GBF, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 3300 Braunschweig, FRG *Zentralinstitut für Molekularbiologie, AdW der DDR, R. Rössle Str. 10, 1115 Berlin-Buch, GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Enzyme sensors for D-glucose, L-lactate and L-glutamine were tested for monitoring animal cell cultures. By coupling with FIA-techniques an online process control could be realized

      Dransfeld, I.; Hintsche, R.; Moritz, W.; Pham, M. T.; Hoffmann, W.; Hueller, J.; Central Institute of Molecular Biology, Academy of Sciences of the GDR, R.-Roessle-Str. 10, Berlin-Buch, 1115 6DR * Humboldt University, Berlin, GDR ** Central Institute of Nuclear Research, Academy of Sciences of the GDR, Rossendorf, GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Enzyme field effect transistors for Qlucose and lactate were developed by coupling a new transducer type, the fluoride ion sensitive FET, with the peroxidase-catalyzed reaction liberating fluoride ions from organo-fluoro compounds in the presence of hydrogen peroxide “which was produced in coupled oxidase reactions. Glucose oxidase or lactate oxidase and peroxidase were coimmobilized with human serum albumin or polyurethan at a multigate pF-FET. 4-fluoroaniline and pentafluorophenol were used as organo-fluoro compounds. The main functional parameters are given.

      Hintsche, R.; Dransfeld, I.; Scheller, Frieder; Pham, M. T.; Hoffmann, W.; Hueller, J.; Moritz, W.; Academy of Sciences of the GDR, Central Institute of Molecular Biology, R.-Roessle-Str. 10, Berlin-Buch, 1115 GDR, Central Institute of Nuclear Research*, Rossendorf, GDR, Humboldt University**, Berlin, GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Enzyme field effect transistors (ENFETS) were assembled by immobilizing enzymes to a multigate proton and fluoride ion sensitive field effect transistor. Two variations of difference mode measurements of glucose have been realized. All the gates and electrodes at the chip have been homogeneously covererd by a crosslinked, enzyme containing Polyurethane matrix. The difference of the output voltages of a PH-ENFET and a pF reference FET (REFET) or the difference of a pF-ENFET and a pHREFET are measured using an integrated thin film noble metal electrode as pseudo-reference.

      Bilitewski, Ursula; Schmid, Rolf D.; GBF - Gesellschaft für Biotechnologische Forschung, Abteilung fir Enzymtechnologie, Mascheroder Weg 1, D-3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      The basis of this alcohol sensor is the oxidation of ethanol by NAD* with alcohol dehydrogenase as the catalyzing enzyme. The NADH formed is detected electrochemically at 700 mV versus a Ag/AgCl-electrode. The electrode used is a carbon paste electrode with the enzyme and the cofactor mixed into the paste. The sensor can be used up to concentrations of approximately 0.15 Mol/1 (i. e. 1 Vol%), with a response time of 15 s and a stability of at least 7 days.
    • Biosensors Based on Chemically Modified Electrodes with Dehydrogenases as Biological Components

      Kulys, Juozas J.; Bilitewski, Ursula; Schmid, Rolf D.; GBF - Gesellschaft fir Biotechnologische Forschung, Abteilung fir Enzymtechnologie, Mascheroder Weg 1, D-3300 Braunschweig Institute of Biochemistry, Lithuanian Academy of Sciences, Vilnius, USSR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Graphite electrodes were modified by adsorption of phenazine methosulphate (PMSt) or N-methyl phenazinium tetracyanoquinodimethane (NMP*TCNQ-) as mediators for NADH-oxidation. Different dehydrogenases were entrapped in a dialysis membrane together with molecular weight enlarged NAD (NAD-PEG). These electrodes could be used for the detection of substrates of dehydrogenases at potentials around 0 mV versus a Ag/AgCl-electrode.

      Kirstein, Dieter; Kirstein, Lore; Scheller, Frieder; 1: Akademie der Wissenschaften der DDR, Zentralinstitut für Molekularbiologie, DDR-1115 Berlin 2: Klinikum Berlin-Buch, Institut für Laboratoriumsdiagnostik DDR-1115 Berlin (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Amperometric enzyme electrodes for NAD(P)H as well as bienzyme electrodes for dehydrogenase substrates have been developed on the basis of the horseradish peroxidase catalyzed aerobic oxidation of reduced pyridine nucleotides. Limits of detection are: 20 ymol/l NADH or 30 umol/1 NADPH in the HRP electrode and 0.8 umol/1 NAD(P)H, 80 pmol/l glucose, 100 ywmol/l ethanol and 200 umol/l isocitrate in HRP-dehydrogenase bienzyme electrodes with cofactor recycling. Relative standard deviations are 4 %, measuring frequencies 6-8 samples/h. Other types of amperometric biosensors are based on the electrochenical hydrazine oxidation. The dependence of the anodic current on the hydrazine concentration at constant pH values was used to determine enzyme activities of human serum and bovine eye lens leucine aminopeptidase (LAP) and of human serum alanine aminopeptidase (AAP). Detection limits were 5 units/l, the correlation of the results in serum with the respective optical method was better for AAP then for LAP. Kinetic constants of bovine lens LAP were found in the same range as with the optical method. At constant hydrazine concentration its oxidation current is a linear function of the hydroxyl ion concentration. This dependence was used to develop an amperomeric urea electrode. Typical parameters are: linear range 0.8-35 mmol/l, response time 20s, relative standard deviation 1%, frequency 40 samples/h and operational stability two weeks. The urea content in pure solutions, in dialysates of artificial kidneys and in human serum was determined in good correlation with Berthelot’s method. Buffer influences were eliminated by two electrode difference measurements.

      Dittmar, K. E. J.; Conradt, H. S.; Hauser, H.; Hofer, B.; Lindenmaier, W.; GBF, Gesellschaft fiir Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      The expression of foreign genes using recombinant DNA technology in various host systems has permitted the production of human proteins of therapeutic interest in high amounts. Manyclinically important human proteins are posttranslationally modified. However, the inability of microbes to perform mammalian-type of posttranslational modifications of proteins is a major shortcoming. Alternative expression systems are insect and mammaliancells. Principle mammalian types of protein modifications are N- and O-glycosylation. Insect cells, fungi and yeasts are unable to perform the same terminal glycosylation reactions on glycoproteins as mammalian cells. Recombinant DNA technology used for the production of pharmaceutically useful polypeptides has mainly been focused on microbial expression systems (bacteria like E. coli, yeast and fungi). The advantage of microbial expression systems is the high amount of expressed protein that can be obtained. The present communication considers aspects of glycoprotein research relevant to the field of biotechnology and protein design. Results are presented that have been obtained by our group during the last four years concerning the expression of the glycoproteins human Interleukin 2 (Il-2) and Interferon-8 (IFN-8) in different mammalian cell lines, the determination of their carbohydrate residues, the effect of site-directed mutagenesis on their carbohydrate attachment sites and the insertion of peptide domains which function as acceptors for carbohydrates.

      Schomburg, Dietmar; GBF(Gesellschaft fiir Biotechnologische Forschung) Mascheroder Weg 1, D-3300 Braunschweig, WEST GERMANY (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Since the first reports on the use ofsite directed mutagenesis in 1982! protein engineering or - when rationally aimed - protein design has been Tecognized as a promising and fascinating field of research in many countries. In Japan (PERI) and the U.S.A. (CARB)researchinstitutes have been founded with the focus on protein design. More and more researchinstitutes in the United States, Canada, Japan and Europe have been Starting broad research projects on protein design (UK: SERC, W. Germany: GBF, EMBL). Possible prospects for applications of designed proteins with modified activities or other new properties are very high,in the areas of pharmacology, enzyme applications in food industry’, waste treatment and chemical synthesis, vaccine design, biosensors etc.*". This conception wasvery clearly lined out in an excellent article by Kevin Ulmerin 19835, Encouraging results have so far been obtained only for a small number ofcases including insulin, proteases and peptidic protease inhibitors, and some others®’. On the other hand many unpredicted andsurprising results of site directed mutagenesis experiments are reported onscientific meetings and in the literature*’, That demonstrates that our methods andtools in thatarea arestill rather crude and urgently require improvement!", Simplecalculations show that the random approach to protein-engineeringis a very slow one. There are 10°* ways to arrange aminoacids in a medium sized protein chain of 250 amino acids. Ca. 10” molecules would form the whole estimated mass ofour universe. But even whenthe information about the seven most important aminoacids is available and only five changes should be tested for each of the positions, about 80,000 different protein-mutants have to be prepared andtested. This implies that a knowledge of the 3D-protein-structure and a good understandingofthe functionactivity relationship is absolutely essential in order to do rational protein-design. Research projects in protein-design require a close cooperation between groupsspecialising in protein-isolation and purification, in fermentation techniques, in genetic-engineering, in DNAsynthesis and protein-crystallography (protein-NMR techniques are being established). This interdisciplinary connection between protein chemists, molecular biologists and stereo-chemists is essential for the protein-design cycle (Fig. 1) consisting of design, cloning, expression andtesting new proteins starting from known ones.

      Hol, W. G. J.; Laboratory of Chemical Physics University of Groningen Nijenborgh 16 9747 AG Groningen The Netherlands (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1988)
      Protein crystallography is currently undergoing a rapid change in many different ways. One development is the explosion of interest by molecular biologists and immunologists since knowledge of protein sequences, obtained via DNA sequencing, is expanding rapidly, but does often not increase immediately insight into the functioning of the protein. Another change is the recombinant DNA technique which make it possible to obtain large amounts of proteins which were previously only available in minute quantities. A third change is the wide-spread awareness that detailed knowledge of wellselected protein structures is a promising starting point for designing new pharmaceuticals and vaccines, for obtaining new proteins via protein engineering techniques and for inspiring synthetic chemists in their biomimetic endeavours. At the same time many technical aspects of protein crystallography are undergoing a rapid development. Someof them will be describedin this paper. Crystal structures of proteins can be obtained currently in two quite different ways: (i) the "multiple isomorphous replacement" (MIR) method [1-3] for de novo structure determinations, often using additional anomalous scattering information (MIRAS) [4,5]; and, (ii) the "molecular replacement" (MR) method [6-8] for solving new structures related to a known structure. We will discuss the steps involved in obtaining high resolution X-ray structures as outlined in Figure 1. A detailed account of these steps can be found in two volumes of Methods of Enzymology [9].

      Katz, Bradley A.; Department of Pharmaceutical Chemistry University of California, San Francisco San Francisco, California 94143 Department of Biomolecular Chemistry Genentech Inc. 460 Point San Bruno Blvd South San Francisco, California 94080 and Research Department Genencor, Inc. 180 Kimball Way South San Francisco, California 94080 present address: Triton Biosciences Inc. 1501 Harbor Bay Parkway Alameda, California 94501 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      The crystal structures of 4 variants of subtilisin, each one containing an engineered disulfide crosslink have been determined. The geometries of the engineered disulfide groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a relationship between their measured redox potentials and their calculated dihedral energies. Disulfide introduction produced cavities in the protein structures. The cavity produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was partially filled by a disordering of nearby Asn117. The cavities were often filled with ordered water molecules that replaced interactions of the removed groups. Molecular modelling provided insight into the location where a disulfide could be incorporated, and into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed that introduction of disulfides into buried hydrophobic regions resulted in long range concerted rearrangements.

      Dittmar, Kurt E.; Woolley, Paul; Gesellschaft fiir biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig #Kemisk Institut, Aarhus Universitet, DK-8000 Arhus C (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      This essay is an attempt to point up the gap between, on the one hand, the methods currently available to the biologist in the laboratory and, on the other, the kind of data that he or she would need in order to characterise genetically engineered proteins of topical biological interest in such a way as to make use of the techniques of protein engineering. Sgren Kirkegaard was Denmark's greatest philosopher, and he was well aware of the fact. One day he reflected: “To be Denmark's greatest philosopher, ah, that is indeed a fine satire.” By this he presumably meant that he was the only one. These words have encouraged us to philosophize a little about the protein engineering cycle, of which our version is shownin Figure 1. We have dissected the cycle according to two principles, information-theoretical (vertical axis) and epistemological (horizontal axis). The cycle starts from a gene and proceeds via expression to the corresponding protein, which we associate with a set of properties by testing or suitable characterisation. The understanding of these leads by way of theory, experience or intuition to a new gene, and thereafter the cycle continues, a process of which we have seen many impressive examples.

      Blundell, Tom L.; Hubbard, Tim; Johnson, Mark S.; McLeod, Alasdair; Overington, John P.; Sali, Andrej; Sutcliffe, Michael; Thomas, Pamela; Laboratory of Molecular Biology, Departmentof Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, England. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Knowledge-based modelling can be envisaged as a number of steps concerned with the establishment and use of rules to generate a model of a protein. One of the most powerful procedures in learning rules is comparison of related Structures either through alignment of sequences to identify conserved residues or superposition of three dimensionalstructures to identify conserved conformations or motifs. Thus the first step in a knowledge-based modelling procedure is the systematic comparison of families of topologically similar structures. This step will lead to the establishment of "equivalences" between the structures compared and to their clustering based on measures of similarity. The second step involves the projection of the results of the comparisons of three dimensional structures down onto the level of sequence. This step establishes rules relating sequence to structure. These can be expressed as consensus sequences - templates - for topologically equivalenced residues, or as key residues in canonical structures, which are then used to align the sequence of the protein of unknown tertiary Structure. The third step uses the rules established in the second step to generate a three-dimensional model.

      Brange, J.; Drejer, K.; Hansen, J. R.; Havelund, S.; Kaarsholm, N. C.; Melberg, S. G.; Soerensen, A, R.; Novo Research Institute, DK-2880 Bagsvaerd, Denmark (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)
      Different wild type insulins have been used for treatment of diabetes mellitus since Banting and Best (1) isolated insulin for the first time in 1921. This injection therapy has saved many lives but has not been able to reproduce the serum insulin profile obtained by physiological endogenous insulin secretion. The requirements of biosynthesis, processing and storage in the pancreas have put severe constraints on the insulin molecule giving it properties which are not necessary for the biological action of the hormone and which limit the possibilities of obtaining adequate metabolic control in diabetics.The self-association to a hexamer, for example, facilitates proinsulin conversion and its subsequent precipitation as crystals in the storage vesicle (2) but is evidently not related to the interaction of insulin as a monomer to its receptor, and this property delays the transport of insulin from the subcutaneous depot to the circulation (3). Thus pancreatie insulin did not evolve for exogenous administration, and with the aim of producing insulin with improved therapeutical characteristics a whole series of human insulin analogues with changed self-association and ligand binding properties has been developed through molecular modelling and recombinant DNA-technology. Most analogues were designed to have less tendency to associate (4) but a few were also created to possess stronger association or metal-binding properties.
    • A ComputationalTool for Structural Biology: Crystallographic Refinement by Simulated Annealing

      Brünger, Axel T.; The Howard Hughes MedicalInstitute and Department of Molecular Biophysics and Biochemistry, Yale Universit, New Haven, CT 06511 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1988)
      Conventional refinementof biological macromolecules involvesa series of steps, each of which consists ofa few cyclesofrestrained least-squares refinement with stereochemical and internal packingconstraints orrestraints that are followed by rebuilding the modelstructure with interactive computer graphics. Duringthefinal stages of refinement solvent molecules are usually included and alternative conformations for some atomsor residues in the protein may be introduced.