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AN OPTICAL BIOSENSOR FOR THE DETECTION OF A BACTERIAL TOXIN
No full textThe EVIA-biosensor (evanescent wave immunoassay) was evaluated for the determination of
botulinum toxin. Three different assay systems were used: an inhibition type assay using
fluorescent labelled antibody, a fluorescent excitation transfer immunoassay with rhodamine
labelled antibodies as quenchers and a sandwich type immunoassay. The inhibition type assay
system proved to be highly suitable for this sensor technique. 200 ng of the toxin could be
detected
Two Examples of Biosensor Systems Based on Flow Injection Analysis
No full textTwo optical biosensor systems will be presented in this paper: one uses immobilized
enzymes and coenzymes, while the other employs immunoreagents. They can be used for
eultivation monitoring of low and high molecular weight components in fermentation broth.
Both sensors are based on the principles of flow injection analysis. The principles of these sensor
systems, their characteristics, and their application will be discussed
ENZYME- AND IMMUNO-FIA-CONTROL OF ANIMAL CELL CULTURES
No full textThree enzyme sensors (for glucose, lactate and glutamine) have been
coupled simultaneously with a flow-injection analysis device on-line
with bioreactors harboring monoclonal antibody and interleukin-2
producing cell lines. Two approaches for FIA-coupled immunosensors
were demonstrated for detection of alpha-interferon
ENZYME IMMUNOASSAYS USING FLOW INJECTION ANALYSIS WITH ELECTROCHEMICAL DETECTION
No full textThe utility of FIAEC for enzyme immunoassaysof low detection limit can be
constrained bythe existence of a nonfaradaic capacitance current that records with the
analytical faradaic signal. Since the magnitudeof this unwanted signal diminishes with
smaller potential, conditions can be found where the interference becomes unimportant.
This was done here by developing p-aminopheny] phosphate as a substrate for alkaline
phosphatase, which yields p-aminophenol as product, oxidizable at between +100 and
+300 mV. Additional advantageis gained by matching the compositions of the sample and
mobile phase solutions. The useof p-aminopheny] phosphate as a substratefor
electrochemical enzyme immunoassay was demonstrated by sensitive assays for
theophylline, IgG and alpha-fetoprotein
SIALYLTRANSFERASES; THEIR SPECIFICITY AND THEIR USE IN CARBOHYDRATE REMODELLING.
No full textIn order to apply sialyltransferases in the remodelling of the
carbohydrate chains on biologically active glycoproteins, it is a
prerequisite to know the fine specificity of these enzymes. In this
report the specificity of several sialyltransferses involved in the
sialylation of O- and N-linked oligosaccharide chains is reviewed.
Also novel results on the branch specificity of a3- and a6-sialyltransferase
are reported.
The potential application of these enzymes in carbohydrate
remodelling was studied using human chorionic gonadotropin (hCG) as a
model glycoprotein. Differently sialylated preparations of this
hormone were obtained and tested for their stimulatory effect on
steroidogenesis in Leydig cells in vitro. Asialo-hCcG appeared to be
only 45% as effective as native hCG. a3-Resialylation of the O-linked
chains on the ß-subunit of this hormone did not restore the
biological activity to a higher level. By contrast, 55% a6-
resialylation of the N-linked chains yielded a preparation which was
almost as active as native hCG. Interestingly, further sialylation by
the a6-sialyltransferase resulted in a decrease of the bio-activity
to levels lower than obtained with asialo-hcG.
It is concluded that the lectin-carbohydrate binding, which is
part of the process that triggers the biological respons of the
target cell can be mimicked by N-linked chains carrying a6-linked
sialic acid. However, too high a density of such residues interferes
with this interaction
REGULATION OF TERMINAL GLYCOSYLATION
No full textMammalian cell lines used for production of recombinant glycoproteins elaborate terminal
glycosylation structures on N-linked and O-linked carbohydrate groupsthat are determined by
the glycosyltransferases expressed by these cells. As many as twelve glycosyltransferase
cDNAs have now been cloned by a variety of strategies (1). By expressing these
glycosyltransferase cDNAs incells not normally expressing them, it is now possible to alter
the cellular glycosylation machinery to produce new terminal glycosylation sequences (2,3).
This principle was demonstrated by expressing the rat B-galactoside 02,6 sialyltransferase
(a2,6ST) cDNA in CHOcells, which are known notto express the product of this
sialyltransferase. After selection for stable expression, these cells were shown to produce Nlinked
carbohydrate groups with terminal 02,6 linked sialic acid, demonstrating an altered
glycosylation machinery (2)
CRYSTALLIZATION AND X-RAY STUDIES OF LIPASES
No full textSeveral structure determinations of lipases are underway in Marseille. Most projects
are at a heavy-atom derivative search stage. These projects belong to three classes:
i. Cutinases are a group of extracellular fungal hydrolytic enzymes capable of degrading
the insoluble lipid polyester matrix, i.e. cutin, which covers the surface ofplants.
Their weight is 22,000 Da, signifantly lower than all other lipases. A recombinant cutinase
from F. solani pisi is expressed and excreted with very high yields in E.coli cultures.
Cutinase was crystallized (PEG 6000 15-20% ‚pH 7.0 to 10.0,20°C) in space
group P2, with cell dimensions 35.1A,67.4A,37.05 A, B=94°. They diffract to 1.5 resolution
(Rsym=4.41%). Data from native and derivatives have been collected. MIR
phasingis in progress.
ii, Gastric lipases are pH resistant enzymes with a molecular weight of ca 50,000, including
15% sugars. Every glyco-variant from different species have been purified by
ief, and submitted to crystallization, using parameters found by an incomplete factorial
analysis method. All attempts were successful, but most crystals were unsuitable to Xray
studies due to huge cell dimensions. Dog gastric lipase crystals, the most suitable
for X-ray study (pH 6.8, 12% PEG 6000, 20°C; P2,2,2 :182A, 211A, 98 A), contain ca
8 moleculesin the asymetric unit , and therefore diffract to only 4A on lab sources.
iii, Crystals of a complex between porcine pancreatic lipase (50000 Da) and its co-factor,
colipase (11000 Da), have been obtained. These 1/1 complex crystals are disordered,
and diffract weakly. Horse pancreatic lipase has been crystallized in space group
P2,2,2,, (89A,97A,145A ; pH 6.0, PEG 8000 10%, 20°C)and a 2.3 A native data set
has been collected. One PCMBS derivative was not sufficient to produce a good map,
and weare still looking for other derivatives. Two crystal forms of human pancreaticlipase
(purified from pancreas juice) have been obtained, different from the one reported
in the litterature. They crystallize in the same conditions and space groups are P2, and
Py. Native data sets have been collected
COMPARATIVE STUDY ON PRIMARY STRUCTURES OF TWO LIPASES FROM GEOTRICHUM CANDIDUM
No full textGeotrichum(G.) candidum produces two extracellular lipases I and II, the lipase I
being the predominant component. The lipase I and II cDNAs were cloned, and their
nucleotide sequences were determined. The nucleotide sequences included the N- and Cterminal
amino acid sequences and the partial amino acid sequences of the lipases.
The mature lipases I and II were of the same length, their overall identity being
84%, Furthermore, four Cys residues were completely conserved, which may participate
in the formation of disulfide bridges. A homology search indicated that the G.
candidum lipases and Candida cylindracea lipase are homologous enzymes, and that they
are members of the cholinesterase family
STAPHYLOCOCCALLIPASES AND PHOSPHOLIPASES
No full textFor many years it has been known that certain Staphylococci produce and release lipolytic
enzymesinto the culture medium. In order to obtain more information about the genetic and
biochemical properties, the lipase genes of Staphylococcus hyicus and Staphylococcus aureus were
cloned and sequenced. The S. hyicus lipase gene was cloned and expressed in Staphylococcus
carnosus. From this organism, the enzyme was secreted into the medium as a protein with an
apparent molecular mass of 86 kD. This protein was purified and the N-terminal sequence revealed
that the primary gene product was processed at the proposed signal peptide cleavagesite. In S.
hyicus, the DNA-donorstrain, the lipase was further processed by an extracellular protease to a 46
kD protein, which exhibited a 3-fold higher specific activity as compared to the 86 kD protein. The
46 kD protein waspurified to homogeneity. The lipolytic activity was dependent upon the presence
of Ca2*. The purified lipase revealed an extremely broad specificity. The enzyme hydrolysed not
only triglycerides, but also naturally occurring phosphatidylcholines and lysophospholipidsto free
fatty acids and water-soluble products. The 641 aminoacid residue S. hyicus lipase is organized as a
pre-pro-lipase, consisting of a 38 aminoacid signal peptide, a 207 aminoacid pro-peptide, and a
396 amino acid mature lipase. Secondary structure predictions revealed that the pro-peptideis
characterized by hydrophilic protein domains, while in the mature lipase, hydrophobic protein
domains are predominate. It was demonstrated by gene fusionstudies that the pro-peptide is
necessary for efficient lipase secretion
Biosensors based on capacitance measurements and 2D-microelectrophoresis on lipid membranes
No full textA great numberofbiological receptor moleculesare located within lipid bilayers. How can one use
them for biosensor applications? Biological receptors should be reconstituted into their natural
environment- e.g. the lipid bilayer - to achieve proper function.
It is therefore necessary to develop (1) methodsfor the preparation of high resistance lipid/protein
membranesonsolid supports and (2) appropriate transducers for the conversion of the chemicalto
an electrical signal. In our case we apply capacitance measurement.
Amplification of the sensor signal is a further important requirement for biosensors to achieve
sufficient signal/noise ratios of the sensor. We suggestthe lateral 2D-microelectrophoresis. This could
be a useful tool for the separation and local accumulation of different receptors