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PURIFICATION AND CHARACTERIZATION OF A MICROBIAL XANTHINE DEHYDROGENASE HIGHLY ACTIVE TOWARDS HXPOXANTHINE
No full textWe purified xanthine dehydrogenase (EC 1.2.1.37) from Pseudomonas putida Fl, which
was screened with hypoxanthine as the main carbon and nitrogen source. The purified
enzyme was homogeneous judged by polyacrylamide gel electrophoresis with or without
SDS and HPLC on a TSK G-3000 SW column. The enzyme showed an absorption maximum at
457 nm in the visible range and shoulders between 300 to 400 mm and 500 and 600 rm.
The absorption ratio at 280 nm to 450 mm was 5.9. The enzyme showed higher activity
with hypoxanthine than xanthine as substrate with NADt as an effective electron
acceptor. Purine was a poor substrate and neither adenine nor guanine was oxidized by
the enzyme. Specific activity was 80.2 U/mg with hypoxanthine as substrate, and Kn
values for hypoxanthine, xanthine and NAD‘ were 40, 64 and 52 UM, respectively. The
molecular weight of the native enzyme was estimated to be 350,000 by HPLC on a TSK G-
3000 SW column, and those of two different subunits were 92,000 and 46,000 by SDSpolyacrylamide
gel electrophoresis, respectively. Other catalytic properties of the
enzyme were also reported
APPLICATION OF A PLANAR ENZYME MICROELECTRODE TO BIOPROCESS MONITORING
No full textUsing microelectronic technology it has been possible to develop disposable glucose
electrodes (1). The transducer is realised using thin-film deposition and lift-off techniques, and
consists of two Pt electrodes (working and counter) and an Ag/AgCl reference electrode. This
transducer is covered with an enzyme membrane. The hydrogen peroxide formed by the
enzyme catalysed reaction is measured at + 0.6 V.
For use in FIA, such electrodes must meet certain specifications as to response time,
sensitivity, reproducibility, accuracy, and electrode lifetime. We have investigated the
performance of electrodes in an FIA system with particular reference to the measurement of
glucose at low concentration in culture media. Typical performance characteristics are -
a) a lifetime of 14 days
b) a sensitivity ( peak height ) of 30 yA M-1 at a flow rate of 1.4 ml min"! and sample volume of
190 ul
c) anoise level below 5 pA
d) a linear response from 1.0 HM to 1 mM glucose
The detection limit is determined not by the signal / noise ratio, but by the presence of
interfering compounds within the sample. We have determined quantitatively the level of this
interference during mammalian cell culture, and have shown it to be much smaller than
expected. It is possible to include additional membrane layers in construction of the sensor.
These would be designed to reduce such interference and thus increase specificity. Our
results suggestthat this may not be necessary
ON-LINE DETERMINATION OF GLUCOSE AND LACTATE CONCENTRATIONS IN ANIMAL CELL CULTURE BASED ON FIBRE OPTIC DETECTION OF OXYGEN IN FLOW-INJECTION ANALYSIS
No full textA flow-injection analysis (FIA) system for on-line monitoring of glucoseandlactate
concentrations in animal cell cultures based onfibre optic detection of oxygen consumption
using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is
described. The consumption of oxygen was determined via dynamic quenchingof the
fluorescenceof an indicator by molecular oxygen. GOD and LODwere immobilized on
controlled pore glass (CPG) in enzyme reactors which weredirectly linked to a specially
designed fibre optic flow through cell covering the oxygenoptrode. The system is linear
for 0 - 30 mM glucose, with a r.s.d. of 5% at 30 mM (5 measurements) and for 0 - 30
mM lactate, with ar.s.d. of 5 % at 30 mM (5 measurements). The enzyme reactors used
were stable for more than 4 weeks in continuous operation, and it was possible to
analyse up to 20 samples per hour. The system has been successfully applied to on-line
monitoring of glucose and lactate concentration of an animalcell culture, designedfor
the production of recombinant human antithrombine Ill (AT-III)
INNOVATIONS IN THE USE OF ENZYMES IN FLOW INJECTION ANALYSIS
No full textSome new approaches to the use of immobilized and dissolved enzymes
in flow-injection systems (FIA) such as their incorporation into fast
detectors, an placement of the enzymatic reactor in an unvival place
such as the injection loop or in the detector flow-cell and the
HPLC-FIA association are presented and discussed. Interesting
determinations of a variety of substrates in real samples have been
developed through these innovations, which offer clear advantages in
terms of sensitivity, selectivity, rapidity, precision, automation,
etc., as compared with conventional FlA-enzyme methodologies
Simulations of the Potential Generation of an Enzyme-pH Fieldeffecttransistor (ENFET)
No full textIn order to verify the chances of the enzyme-pH
fieldeffecttransistor (ENFET) for the detection of urea
in practical applications, in our group computer
simulations have been carried out that scrutinized the
influence of various parameters on the measurement
signal.
The described sensor uses a glutardialdehyde
membrane containing the enzyme urease and an ISFET
transducer, and is immersed in a buffer solution. If urea
is added to the solution, it is metabolized to carbon
dioxide and ammonia. The resulting pH-increase in the
The program calculates a local distribution of substrate
concentration and pH-value in the membrane. The
influence of the above mentioned concentrations on the
pH-value (which can be regarded as the measurement
signal) is discussed.
Above all these simulations show that for a working
enzyme-pH sensor a well defined composition of the
sample solution is essential. Therefore the ENFET
works best in a flow-through-system, where all
parameters can be kept constant, or together with a
microtitration unit.
membraneis measured by the ISFET.
The simulation program is based on Michaelis-Mentenrelations
and on adequatediffusion-reaction-equations
for the concentrations of the enzyme, the substrate, and
the reaction products, as well as the buffer capacity.
Also the thickness of the membrane is taken into
account
BIOSENSORS WITH OXIDOREDUCTASES AND INTEGRATED COENZYME OR MEDIATOR RECYCLING
No full textMost amperometric enzyme electrodes demand integrated coenzymes and/or mediators for the
transfer of redox equivalents between protein and transducer. Thefree diffusibility required for
this function of the mediators implies simultaneously their leaking from the electrode chamber.
Approaches to overcomethis problem, aiming on the construction of compact independent oxidoreductase
electrodes are reportedin this contribution.
first attempt is based on the covalent binding of oxidoreductases on various conducting supports.
Independent from the material and the binding method no direct electron transfer was obtained
in any case. A subsequent immobilization of mediators through long spacersis therefore
envisaged. In another approach, the functionalization of conducting surfaces with orientated
mediators boundthrough conducting spacersis investigated, aiming on the subsequentaffinant
binding of coenzymes. The most successful and promising way seemsto bind mediators or
coenzymes through spacers to the enzymes themselves and to immobilize these complexes onto
or within functionalized electrode surfaces
NEw MODIFIED ELECTRODES FOR ELECTROCATALYTIC OXIDATION OF NADH BASED ON CONDUCTING POLYMERS
No full textThe dehydrogenases are an importantclass of oxidoreductase enzymes, which depend on
the cofactors B-nicotinamide adenine dinucleotide (NAD+)or B-nicotinamide adenine
dinucleotide phosphate (NADP+)as electron acceptors. These enzymes catalyse the oxidation
of specific substrates, with concomitant production of the reduced cofactor,
NAD(P)H. In order to develop amperometric biosensors for determination of such sub-
Strates, much researcheffort has been directed towards the electrochemical oxidation of
NADH. However, direct oxidation of NADH requires a large overpotential (1) andis thought
to involve radical intermediatesthat mayresult in electrode fouling (2)
BIOSENSORS FOR FERMENTATION CONTROL
No full textThis project involves the on-line determination of substrates/products and cell mass of microbial
bioprocesses and the substrates/products of animal cell cultures. The strategy adopted is the
development of a flow injection analysis (FIA) system used for the simultaneous detection of a
number of parameters from a single cell-free sample stream and the on-line detection of viable
biomass.
The manuscriptis limited to a critique of the choice of parameters to be measured, developmentof
biosensors/ biochemistry based analysis systems andtheir incorporation in FIA
DETECTION OF LIGANDS VIA BACTERIAL LUCIFERASE
No full textBacterial luciferase is a widely applied enzyme in medical diagnosis. The applications
harness the FMNH,-dependency of the luciferase. Via FMNH,it is possible to couple
other FMN-dependent enzymes or enzyme systems. With the help of FMN/NADHoxidoreductase
coupling with NAD/NADH-dependent enzymes and enzymesystems it
is possible to broaden the range of application”.
luciferase
FMNH,+ O, + aldehyde ------------------ » FMN + H,O + acid + light
The amount of emitted luminescent light is proportional to the substrates and can be
easily monitored with a luminometer. In addition to FMNH,, other substrates are
converted, for example an aldehyde into the corresponding acid.
Very low concentrations of these aldehydes can be detected and the reaction has a large
linear range (5 to 100000 nM). Maximal activity (luminescence) is achieved through nalkanes
with a chain length of C-10 to 14.
Hereit will be shown that modified aldehydes are as effective substrates as unmodified
ones. Furthermore, the interaction of both will be characterized and a general scheme
of ligand detection presented
APPLICATION OF NADH OXIDASEIN FIBRE OPTIC BIOSENSORS
No full textA 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