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CRYSTALLIZATION AND PRELIMINARY X-RAY ANALYSIS OF A LIPASE FROM A SPECIES OF PSEUDOMONAS
No full textA lipase from a species of Pseudomonas has been cloned and expressed
in E. coli. Variants of this lipase have been generated, using sitespecific
mutagenesis, that have significantly altered k,,,, K, and
substrate specificity. We have undertaken to determine the threedimensional
structure of this enzyme using X-ray crystallography.
Crystals have been obtained and these crystals diffract to 2.5 Angstrom
resolution. We are now in the process of evaluating heavy atom
derivatives to be used to improve the phase information used to calculate
electron density maps
A MOLECULAR VIEW OF LIPOPROTEIN LIPASE AND HEPATIC LIPASE STRUCTURE AND FUNCTION
No full textLipolytic enzymes play a pivotal role in the metabolism of triglyceride-rich lipoproteins
circulating in plasma. The primary structures of many lipases have now been elucidated by
molecular cloning of their cDNAs. To gain further insights into the molecular biology of lipoprotein
lipase (LPL) and hepatic lipase (HL), we have determined their genomic organization.
Both enzymes are members of a lipase gene family. Based on information derived from the
genomic structures it is now possible to directly assess the molecular basis of familial LPLdefiency,
a rare disorder of lipid metabolism involving the massive accumulation of chylomicrons
in the plasma. Employing gene amplification techniques with LPL exon-specific oligonucleotide
primers and direct DNA sequence determination, we have characterized a kindred with
classical familial LPL-deficiency. Loss of LPL enzymatic activity was found to be caused by an
amino acid substitution close to the putative active site
USTILAGO MAYDIS LIPOLYTIC ENZYMES: CHARACTERIZATION AND PARTIAL PURIFICATION
No full textThe crude lipase preparation of Ustilago maydis ATCC 14826 (after
growth on coconut oil) was studied with respect to hydrolysis and
esterification potential, resp., as well as to purification of lipolytic
enzymes. Concerning substrate specifity (hydrolysis) among
triglycerides short chain substances were cleaved to an higher extent
than long chain or unsaturated compounds. Obvious inhibition of lipase
activity was observed when additional amounts of linoleic or linolenic
acid were used during hydrolysis of coconut oil.
After Amberlite XAD-2 immobilization and transfer into n-hexane the
wax ester synthesis potential was confirmed.
The purification of crude lipase preparation by chromatographic methods
led to two lipolytic enzymes
Title_Preface_Contents_List of authors_Photo of the participants
No full textSince the last German status seminar on biosensors was held at the GBF in May, 1989, with many
participants from the German-speakingcountries (Austria, Switzerland and, at that time, the German
Democratic Republic), Germany has becomeunited. This alone was reason enough to organize, for
the first time, a true "All-German Biosensor Meeting" - a task undertaken jointly by the Gesellschaft
fiir Biotechnologische Forschung (GBF) in Braunschweig and by the Zentralinstitut fiir Molekularbiologie
(ZIM) in Berlin-Buch, with the support of the Bundesministerium für Forschung und
Technologie (BMFT) [the National Ministry of Research and Technology, Bonn].
As a format, the organizers chose not only to invite as many researchers in the field of biosensors
from Germanyas possible but also to expand the perspective of the meeting by inviting top-experts
from the international biosensor community. As a result, almost 150 scientists from German institutes
and companies and 15 foreign experts from Austria, Bulgaria, France, Japan, the USSRandits
memberstates, the United Kingdom, the United States of America, Sweden and Switzerland gathered
from May 12 to 14 at the Bogensee Center near Berlin.
The presentations included different types of biological recognition elements (e.g. enzymes, microbes,
antibodies, receptors and lipid membranes), as well as a broad range of transducers (e.g. electrodes,
optodes, FETs and piezoquartzes).
During the second day of the meeting, international developments in the field could be overviewed
thanks to the presentations of leading foreign experts. Finally, in a farewell meeting at the Walther-
Nernst-Auditorium of the famous Humboldt University, a historic outline of this famous place, kindly
offered by Prof. H. Bartelt of the Humboldt University, was followed by remarkable lectures from
Prof. I. Karube of RCAST Tokyo andProf. P. Fromherz of Ulm University.
The presentation of work during the conference wasstructured according to the regional activities in
biosensor R & D throughout Germany. The editors have chosen to stick to this schedule since it
allows easy identification of the projects of each individual group. Tofacilitate understanding for the
non-German readers of these proceedings, the locations of the laboratories present at the meeting
are indicated in Fig.1.
The organizers of the meeting who are also the editors of this monograph wish to express their
gratitude to the BMFT and the GBFfor their very significant financial Support, as well as to many
staff members of the GBF and the ZIM, of which the names of Birgit Balster, Heidi Rabe, Silvia
Schmidt, Margit Henselmann, may serve Just as examples. Their tireless help madeit possible to
Prepare and managethe conference successfully. Further thanks are due to Dr.J.-H. Walsdorff as the
copy editor of this monograph, and to Ms. Doris Perl for carefully editing the manuscripts
ORGANIC PHASE BIOSENSORS
No full textTwo types of amperometric enzyme electrode for use in organic solvents are devised. One of them
incorporates horseradish peroxidase for the determination of hydrogen peroxide in organic media.
The enzyme was co-adsorbed with an electron mediator, potassium ferrocyanide, on the surface of
a graphite foil electrode, making reagentless measurementpossible. The electrode can be operated
in dioxane, chloroform and chlorobenzene, the presence of a small quantity of aqueous buffer being
essential for sensor activity. During two weeks of intermittent use the sensitivity of the electrode
decreases by 60 %. At least 50 assays can be performed with a single sensor. As an alternative approach
to assemble oxidase sensors for use in organic solvents, tyrosinase was adsorptively coupled
to a dialysis membrane combined with a conventional oxygen probe. The sensor was successfully
employed for the direct determination of phenolin chloroform
A Microbial Sensor for BOD
No full textEiochemical oxygen demand (BOD) is a widely used parameter for the
determination of biodegradable organic compounds in waste water. The
conventional BOD test takes 5 days and is unsuitable for process
control. A more rapid estimation of biodegradable organic compounds is
possible by using a microbial sensor containing whole cells immobilized
on an oxygen electrode.
The first report of such a microbial BOD sensor was published in 1977 by
Karube et al. The number of such biosensors is growing. BOD sensors have
been developed using the following microorganims: activated sludges
obtained from waste water treatment plants (Karube et al. 1977, Strand
and Carlson 1984), Trichosporon cutaneum ((Hikuma et al. 1979, Harita et
al221985, Riedel et al. 1988,1990), Hansenula anomala (Kulys and
Kadziauskiene 1980), Clostridium butyricum (Karube et al 1977), and
Bacillus subtilis (Riedel et al. 1988)
INTEGRATED ENZYME THIN FILM METAL ECTRODES
No full textMiniaturized electrodes have been constructed as transducers of biosensors. Onto
oxidized silicon wafers Ti as adhesion and Au as conductivity layers have been deposited. A
120nm Pt layer forms the upper layer for amperometric and impedimetric electrodes, whereas
Sb/SbO or Ag/AgCl composite layers are the electro-chemically active layers of the
potentiometric and the reference electrodes, respectively. They can be combined in different
shapes and sizes at the chips and applied in test strips or flow through devices.
The amperometric electrodes with a upper Pt layer are stable at potentials of water
electrolysis. The reference Ag/AgCl composite electrode is more resistent against light and
dissolution than the common surface bound AgCl layers at silver.The pH dependency of the
Sb/SbOelectrode is 56 + 0.1 mV/pH and was observed to be stable over a period of about 30
days, which should be useful for the normal working time of biosensors
FIRST STUDIES ON THE DEVELOPMENT OF A BIOSENSOR FOR TRIGLYCERIDES
No full textA new method for triglyceride analysis is described. It is based on the
fluorescence measurement of NADH, which is obtained in a coupled enzymic
Process, i.e., hydrolysis of triglycerides by regiounspecific lipase and
subsequent oxidation of glycerol by NAD+ dependent glycerol
dehydrogenase. The emerging fluorescence signal at 465 nm following
excitation at 365 nm could be monitored also in turbic solutions, thus time
consuming pretreatments such as extractions become obsolete. The method
was also applied in a FIA-type arrangement
DEVELOPMENT AND EVALUATION OF BIOSENSORS FOR HIV-SEROLOGY
No full textDuring the last decade a new generation of sensors,
the biosensors, has been developed for the quantitative
measurement of biological substances. Application
and specifity of a biosensor are determined by
the recognizing biological/biochemical layer and the
type of transducer. The specific interactions between
enzymes and their substrates, antibodies and antigens
or between receptors and hormones are responsible
for the selective binding of biological components in
solution.
In serological diagnosis the rapid and sensitive detection
of a viral antigen or the induced immunological
response is ofvital interest. Other fields, such as the
utilization in production and control processes, extend
the spectrum of possible applications for such sensor
systems.
The HIV-system has been chosen as a model system.
A peptide antigen from the p24 core protein and mu-
rine monoclonalantibodies were used forinitial experiments.
Experiments under morerealistic conditions
were performed with a consensus peptide from the
hypervariable V3-loop of the HIV-virus and the reactive
rabbit immunosera.
Ionsensitive fieldeffect transistors and piezoelectric
erystals were tested with respect to their suitability for
the detection of antibodies. These immunosensors
have to be evaluated and optimized with regard to
their turn-over periods, sensitivity and cross-selectivity
underexactly defined conditions,
Theuse of a sensor system based on a biosensor generally
possesses - in comparison to conventional
analytical systems - advantageslike reduced influence
of environmentalinterferences, short response times,
and the possibility to develop “intelligent sensors" with
the help of integrated signal processing
BIOSENSORS BASED ON RECEPTORS : OPTICAL TRANSDUCER PRINCIPLES
No full textOptical spectroscopy is a non-destructive method for characterizing biological layers and
surfaces as well as for monitoring changes in concentrations of an analyte interacting with the
layers (23,24,25,26). Knowledge about stability, thickness, and structure of the layer are prerequisites
in the intention to optimize such systems with respect to biosensorfeasibility. Therefore,
lipid bilayers using lactose-permease as a transport protein (s. chapter 1) and synthetic
lipopeptide-antigens (s. chapter 2) were examined. Spectral ellipsometry (27) yields information
about the interaction of polarized light with the biological membranesin addition to measurements
of capacitance, conductivity, and molecular properties (s. chapter 4). Fluorescence and reflectance
measurements dependent on time allow to monitor the dynamic processes of fluorescence
quenching of a labelled antigen by a specific antibody. Furthermore, the interference of
reflected beams can be applied to the examination of immuno-sensors. Spectral detection increases
the amountof information and reduces the danger of artefacts. However, multi-wavelength
detection by diode arrays lowers sensitivity and increases the experimental problems