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A NOVEL CHOLESTEROL ESTER HYDROLASE FROM PSEUDOMONASSPEC
No full textThe genefor a cholesterol ester hydrolase (CE) [E.C.3.1.1.13] of a Pseudomonas spec. strain was
cloned in pBR322 by screening a plasmid library with synthetic oligonucleotides derived from tryptic
fragments ofthe purified enzyme. Subsequent sequence analysis of two overlapping clones showed a
large open reading frame of 0.9 kb predicting a protein with a molecular weight of 30.700 kD. It
contains a consensus-like signal peptide (SP) of 24 amino acids and a conserved sequence around the
putative active center which is homologousto that of some other prokaryotic as well as eukaryotic
esterases.
No enzymatic activity as well as no immunological reaction could be detected in E. coli cells
harbouring the CE-gene with 1.5 kb of its own upstream sequence on high copy plasmids. In order to
improve gene expression the CE-gene was cloned behind strong E. coli promoters, the translation
initiation region was optimized and several modifications of the SP-sequence were tested. The amount
of CE after induction was 5 to 10 % oftotal protein but all of the enzyme was present in inclusion
bodies. Again no enzymatic activity could be detected neither inside the E. coli cells nor in the culture
medium. As the production of an enzymatically active CE in E. coli was not feasible, the whole
expression cassette including promoter, SP and CE-gene was cloned into mobilizing vector pBT306-1,
a vector system compatible for gram-negative organisms. Transconjugants with this vector in a CEnegative
Pseudomonas strain showed fivefold higher CE-activity than the original strain. Using the
same plasmid no activity could be observed in Pseudomonas putida strain 2440. As CE is a lipoprotein
we assumethat specific lipids are necessary for enzymatic activity
POLYPYRROLE GLUCOSE OXIDASE ELECTRODES: SUPPRESSION OF COOXIDIZABLE SUBSTANCES
No full textWith a polypyrrole glucose oxidase electrode, the analyte glucose can be determined by the
anodic oxidation of the enzymatically produced hydrogen peroxide (H202). Attne high
potential (650 mV vs. Ag/AgCl) necessary for the detection of H20O2 other cooxidizable
substances present in the sample can interfere, resulting in a signal for glucose, that is too
high. In order to suppress the interference, we studied the molecular "sieve effect" (1,2,3) of
the polypyrrole immobilization matrix on a platinum substrate (d=imm). Twodifferent types
of enzymeelectrodes were used (Fig.1)
RAPID DETERMINATION OF BIOMASSACTIVITY BY FLOW INJECTION ANALYSIS
No full textA 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
PREPARATION OF A NADH OXIDASE FOR BIOSENSOR APPLICATIONS
No full textA NADHoxidase from Thermus thermophilus HB8 was purified to homogeneity by a procedure
that is easy to scale up.
The hydrogen peroxide forming NADH oxidase was found to be a monomerof 26 kD by SDSPAGE,
oxidation of NADH (NADPH) occuredin the presence of O, and either FMN or FAD. These
cofactors also enhancedthe stability of the enzyme towards higher temperatures and extreme pH.
The properties of the NADH oxidase are discussed in respectofits applicability in biosensor
techniques
ENZYME ELECTRODE FOR THE DETERMINATION OF L-LYSINE
No full textAn enzyme electrode for the determination of L-lysine is described.
The electrode is based on the catalytic action of a L-lysin.~-oxidase
from Tbichoderma viride i4 which converts L-lysine, Oo and water into
2-0xo-6-aminocaproate, NH3 and hydrogen peroxide. Two of the reaction
partners, 02 and H202, can easily be detected by electrochemical methods
and offer the opportunity to install a sensitive and specific determination
of L-iysine for which other reliable or low cost methods do not
exist.
The enzyme was isolated from the culture extracts of Trichoderma and
purified according to a newly developed method described elsewhere (1).
Compared with the purification scheme for a similar enzyme from Trichoderma
viride Y244-2 this procedure is a different approach. It makes
use of the enzyme*s unusual stability in the pH-range near its isoelectric
point of pH 4.3. It provides a highly purified protein (homogeneous
in 15 % SDS géls) with specific activities of 90 U/mg of protein and
yields of more than 50 %. Only two steps are necessary to purify it from
the bulk material of the culture extracts.
To prepare it for the electrode bhe enzyme is precipitated from its
aqueous solution by the addition of acetone, collected by centrifugation
and fixed on tovthe membrane by a recently developed polymerization
protocol.
First measurements with the lysine oxidase electrode have demonstrated
a linear relationship between L-lysine concentrations and the signal
output. A comparison between determinations of L-lysine with the electrode
and determinations done with an amino acid analyzer indicated no
significant difference between the two methods
Ionsensitive Field-Effect-Structures with Langmuir-Blodgett Membranes
No full textThe electrolyte/insulator/silicon-structure is well suited to investigate and to optimize
membranes for an application in ion-sensitive-field-effect-transistors (ISFETs). Examples for
new membrane materials for chemical sensors are offered by special Langmuir-Blodgett (LB)
films, which show very good properties with respect to long-term stability and sensitivity. An
indispensable condition for the use of the insulator as a carrier for different sorts of
chemically sensitive groupsis its protection against water. Membranes made of a synthetic
rodlike Phthalocyaninato-Polysiloxane (PcPS)-Polymeror a special Polyglutamate (PG) fulfill
this demand. They both exhibit lifetimes of more than 6 months in permanent contact with
electrolyte. While the PcPS-membranes show a pH-sensitivity depending ontheir thickness,
the PG membranes show a much reduced pH-sensitivity. The latter can thus be used for a
reference-element. There are two waysto obtain ion-sensitive LB-materials: Covalent fixing
of ionophores like crown ethers or physical mixing of special sensitive groups to the
polymers. In this paper an Ag*-sensitive membrane with fixed olefin-groups and a K*-
sensitive membrane with a mixed film of PG and Valinomycin are presented
BIOCHEMICAL CHARACTERIZATION OFA HIGHLY SPECIFIC TRIMETHYLAMINE DEHYDROGENASE SUITED FOR THE APPLICATION IN BIOSENSORS
No full textA strain of Paracoccus possessing a NAD (P)*-independent trimethylamine
dehydrogenase could be isolated by enrichment cultivation. The
enzyme was purified up to 4.2 U/mg. Phenazine ethosulfate acts as an
electron acceptor, other artificial mediators such as methylene blue
or K3[Fe(CN)¢] are inactive. Important biochemical data are: pH-optimum:
9.0; substrates: specific for trimethylamine; dimethylamine,
methylamine or $rimethylamine-N-oxide are not converted; Ky(trimethylamine):
6.6*¢10°° M. The calibration curve for trimethylamine (aqueous
solution) using a kinetic photometric assay with dichlorophenol-indophenol
shows a linearity between 0.1 - 0.8 uM trimethylamine
AMPEROMETRIC DETECTION OF LACTATE: A COMPARISON BETWEEN MEDIATED AND PLATINISED CARBON ELECTRODES
No full textTwo electrode configurations,for the amperometric determination of lactate, were
investigated using the enzymelactate oxidase. One system was based on theuse of
mediators (1,2-dimethylferrocene or tetrathiafulvalene) and the other system
incorporated a platinised carbon electrode. Overall the platinised carbon electrodes
proved to be the most stable sensors. During a period of 136-days the electrodes
maintained their range, linearity and response times. The electrodes retained a low
sensitivity for ascorbic acid. Further optimisation and incorporation of the electrodeinto
a flow injection analysis system is now being carried out
EXTRINSIC FIBER OPTIC BIOSENSORS WITH LIGHT EMITTING ENZYME SYSTEMS
No full textA multipurpose fiber-optic sensor was designed in our group, with chemi- or bioluminescent
enzyme systems boundonto a preactivated nylon membraneattached to the end of 8-mm diameter
glass fiber bundle. This waveguide was connected to the PMTof a luminometer enabling remote
detection ofspecific target analytes. The fiber-optic sensor could be used for H7O7 measurements with
immobilized peroxidase. The maximum lightintensity was obtained within 1 min and measurements
could be performed in the range 2 x10°8 M - 2 x10-5 M. With immobilized firefly luciferase, ATP
measurements could be performed over a wide linear range from 2 x 10-11 Mto 1 x 10-6 M.With the
bacterial oxidoreductase-luciferase system, the linear dynamic range was comprised between 1 x 10-9
M and 3 x 10-6 M. Theuseofthe biosensor has been extended to other analyteslike sorbitol, ethanol
and oxaloacetate using specific dehydrogenases co-immobilized with the bacterial enzyme system. The
alternate determination of ATP and NADH with the samesensor using the two co-immobilised
bioluminescent systems on the same membrane was also developed using reaction mediums compatible
with both the activity and stability of each enzyme. The combination of FIA with these biosensors has
also been explored and a reagentless biosensor with co-reactants embedded andreleased in the
immediate vicinity of the sensing tip is now under development
Simulation and Animation of Intracellular Diffusion
No full textA computer program was created which allowed for the calculation and animation of
intracellular diffusion of molecules which diffused from the external space of tissue into the
internal space where they were distributed. Time and spatial distribution of the molecule
concentration could be displayed on a PC screen. The program wasused for the interpretation
of lidocaine effects on the sodium channels of the membraneof spinal sensory ganglioncells