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EXTRACELLULAR LIPASE OF PSEUDOMONAS AERUGINOSA
No full textLipase of Pseudomonas aeruginosa was excreted in form of high M,-
aggregates consisting of protein and lipopolysaccharide. Solubilization
and isoelectric focusing in the presence of the zwitterionic
detergent CHAPS yielded in an electrophoretically pure lipase protein
of M, 29 kDa with an isoelectric point of 5.9. Charge shift electrophoresis
showed that lipase was an amphiphilic protein, its activity
was dependent on the presence of detergent. Lipase cleaved a variety
of different substrates showing no positional specificity. The lipase
gene was cloned and sequenced revealing the lipase consensus sequence
Gly-His-Ser-His-Gly. Polyclonal antibodies were raised against purified
lipase having a titer of 1400 and a detection limit in the picogram
range
PROPERTIES OF HOLOGRAPHIC MEDIA CONTAINING PURPLE MEMBRANE FROM HALOBACTERIUM HALOBIUM AND ITS FUNCTIONAL VARIANTS
No full textPolymeric films of purple membrane (PM) from Halobacterium halobium,
which contains the photochromic protein bacteriorhodopsin (BR) are well
suited reversible media for holographic recording. The sensitivity
(19 cm?/J), resolution (> 5000 lines/mm) and large spectral bandwidth
(400 - 700 nm) of such films are absolutely comparable to the corres- ponding datas of the best known conventional photochromic materials for
holography. By the development of a mutagenesis/selection procedure
which allows to generate and isolate mutants of BR with modified photochemical
characteristics, new possibilities raised to design bacteriorhodopsin
based holographic recording media which cover the physical
demands for different holographic applications like holographic storage
and pattern recognition
BIOEXPERIMENTS WITH IMPROVED CHEMOSTAT METHODOLOGY
No full textDevelopments in bioprocess control include improvement of online
measurements and control. Accuracy and precision of control or availability of
the chemostat systems have reached a very high level. Measurement and
control is based on new software concepts providing automatic control of
complex physiological investigations or bioprocess development work.
Results from cell cyele studies on yeasts synchronized growth, repetitive batch
methods, medium design and integrated process layout for the production of
rhamnolipids and enantiomerically pure compounds are reported.
It is concluded that the breakthrough described in combination with
FIA-coupling are the prerequisites for in vivo studies of the livingcells
ELIMINATION OF INTERFERENCES IN GLUCOSE DETERMINATION IN BLOOD SERUM USING FLOW-INJECTION AMPEROMETRY
No full textA flow-injection system with anodic amperometric detection for glucose
determination in human blood serum samples was optimized by a comparison
of various arrangements of enzyme immobilization and elimination of
interferences such as ascorbic acid, urea and uric acid. The best results
were obtained using a compact membrane biosensor in wall-jet flow-through
detector, where Pt disc electrode was covered with evaporated Nafion layer,
then a polyester membrane with immobilized glucose oxidase and another
protective polyester membrane. In the optimized conditions a linear
response up to 25mM glucose was observed with detection limit 200uM glucose
and sampling rate 120 hr-1 for 20p1 sample volume. Results of flow
injection glucose determination in human serum samples were compared
with Beckman Glucose Analyser 2 and Kone Dynamic analyser
SIMULTANEOUS DETERMINATION OF MULTICOMPONENT IN FOOD BY AMPEROMETRIC FIA WITH IMMOBILIZED ENZYME REACTORS IN A PARALLEL CONFIGURATION
No full textA simultaneous determination system for food components with FIAenzyme
reactor methodology has been developed. The main flow lines of
the system are composed of plural independent lines and plural immobilized
enzyme reactors set in a parallel configuration. Enzymes used
were glucose oxidase for glucose, lactate oxidase for lactate, alcohol
oxidase for ethanol, g-fructosidase, mutarotase and glucose oxidase for
sucrose, fructose-5-dehydrogenase for fructose, sulfite oxidase for
sulfite, and glycerol dehydrogenase for glycerol.
First, simultaneous determination of lactate, glucose and ethanol in
alcoholic beverages and serum was performed by monitoring hydrogen
peroxide. Each corresponding oxidase of the substrate was immobilized
on an Amino-Cellulofine support. Interferences of ascorbate and/or
urate in a sample were completely eliminated by using ascorbate- and
urate-eliminating reactors. Next, glucose, fructose and sucrose were
determined simultaneously. Hydrogen peroxide (for glucose and sucrose)
and hexacyanoferrate (II) (for fructose) were monitored. Sucrose determination
was performed with a glucose-eliminating reactor which was set
just before the sucrose reactor. Interference of ascorbate was also
eliminated by an ascorbate-eliminating reactor. Finally, sulfite,
glucose, glycerol and ethanol in white wine were determined simultaneously.
For the glycerol determination, the NADH produced was monitored
at +0.75 V vs. Ag/AgCl. For sulfite determination, a platinum
electrode covered with dialysis membrane was used in order to diminish
the interference of polyphenol compounds
Flow Injection Immunoanalysis (FIIA) - A New Format of Immunoassay for the Determination of Pesticides in Water
No full textRecently, the appearance of pesticides in groundwater has caused considerable
problems in the control of drinking water quality. Regulations
governing pesticide concentration in drinking water are very
harshly enforced with a maximum allowable pesticide concentration of
0.1 ug/l per pesticide. Approximately 300 pesticides are permitted in
the F.R.G. and their detection at this critical concentration is extremely
difficult. The methods used in water supply stations to control
the pesticide concentration are HPLC and GC/MS. These methods are
time consuming and expensive.
As a result, immunoassays for pesticide detection are of increasing
interest, because they are sensitive, specific, fast and inexpensive
/1/2/3/4/.
Immunoassays are mostly carried out on microtiter plates and a lot of
pipettin is involved. To improve automation we developed a new form of
heterogeneous enzyme immunoassay, named flow injection immunoanalysis
(FIIA), a technique that uses a flow injection system /5/
Title, Preface, Content, List of authors
No full textGlycoproteins (i.e. proteins containing covalently bound carbohydrate) are ubiquitous
constituents of all living organisms including bacteria. The posttranslational modification of
polypeptides with carbohydrate groups is very commonforsecretory as well as integral membrane
proteins of higher organisms which may function as enzymes, antibodies, hormones,
structural or carrier proteins and receptors.
Overthe past two decades theprincipal biosynthetic pathwaysleadingto thefinal carbohydrate
structures of glycoproteins have been elucidated. The introduction of improved techniquessuchas
high-resolution NMRandfast atom bombardment mass spectrometryas well as
the introduction of novel chromatographic techniques for oligosaccharides over the past decade
have expanded our knowledge of the enormous microheterogeneity of oligosaccharide
structures that can be present at even a single glycosylation domain. The biological
significance ofthis structural diversity seen in glycoproteinsis unclear.
Recombinant DNAtechnology has permitted the efficient production of manybiologically
important glycoproteins (membranereceptorsas well as their ligands) by expressionin heterologous
mammaliancell lines. By using defined glycosylation mutantcell lines as hosts as have
been derived from CHOand BHKcells (see paper byP. Stanley, this volume)it should be possible
to obtain pure glycoproteinsof defined carbohydratestructures. Thestudyof the biological
functionality of these glycosylation formswill considerably increase our understanding of
the role of protein linked oligosaccharides.
Pharmaceutical companies’ interest in the productionof clinically important humanproteins
(many of which are glycoproteins) by biotechnological means, will undoubtedly have an
impact on the developmentof glycoprotein biochemistry in the near future. The efforts of the
pharmaceutical industry are directed toward human medicine, and manyclinically useful glycoproteins
(immune-modulators, differentiation factors, glycoprotein hormones and receptors)
are now available from recombinant sources. They shouldbe usedto develop our understandingofbiological
phenomenaassociated with protein linked carbohydrates. However, only
a multidisciplinary approachincluding molecular structure research, computer graphic model
building as well as genetic engineering andcell biologyis likely to be successful.
The present volume evolved from a workshopheld at the GBF in Braunschweigin June 1990
with the aim ofbringing together a balanced mixture of people from university settings whose
interest runsfrom basicscience to the possible practical application of their research, i.e. including
researchers from industrial laboratories with strong biotechnological interest.
I thank the GBF administration, especially Sabine Peters, for help in running the workshop.
Myspecial thank goesto all speakers, chairpersons and contributors to the book. The professionalhelp
of Dr. J.-H. Walsdorff in editing this volumeis gratefully acknowledged
RELEASE OF OLIGOSACCHARIDE-PHOSPHATE DURING THE N-GLYCOSYLATION PROCESS : A ”BY-PASS” IN THE DOLICHOL CYCLE
No full textWe have previously described that N-glycosylation of proteins was
accompanied by the release of oligosaccharide-phosphates and neutral
oligosaccharides, both originating from lipid intermediates. In
order to get a better understanding of the metabolic relationships
between these oligosaccharide species, we have examined the
synthesis and fate of lipid intermediates ina simpler biological
system: a glycosylation mutant of CHO cells which does not
synthesize Man-P-Dol (B3F7 cell line).
B3F7 mutant CHO cells were incubated with tritiated mannose at low
glucose concentration and in the presence of glucosidase and
mannosidase inhibitors (castanospermine and deoxymanno jirimycin
respectively). After extraction and purification, the glycan
moieties of glycoproteins, lipid intermediates, oligosaccharidephosphates
and neutral oligosaccharides were analyzed by HPLC. It
iinteresting to note that oligosaccharide-phosphates were not
glucosylated as was the pool of oligosaccharide-PP-Dol and that, in
contrast, neutral oligosaccharides were glucosylated as were the
glycan moieties of newly glycosylated proteins.
These results indicate that oligosaccharide-phosphates originate
from the cleavage of the pyrophosphate bond of non-glucosylated
oligosacharide-PP-Dol. The glucosylation of lipid intermediates
channels them through the glycosylation of proteins and through the
formation of neutral oligosaccharides which could be, at some steps,
related to the protein glycosylation process itself,
Thus, the cleavage of non glucosylated lipid intermediates into
oligosaccharide-phosphates represents a “by-pass” which allow direct
regeneration of P-Dol. This “by-pass” may control the availability
and the structural suitability of lipid intermediates for protein
glycosylation
GLYCOSYLATION OF A PROTEIN PRECURSORIN YEAST: SECRETION AND PROTEOLYTIC PROCESSING EFFICIENCY
No full textThe 65aa peptide hirudin (rHV2Lys“), which is synthesized in the salivary glands of the
leech Hirudo medicinalis, has been secreted from a transformed Saccharomyces cerevisiae strain
expressing a MFal::rHV2Lys” chimeric gene. The ‘prepro’ part of the protein precursor is
derived from the MFal gene providing an homologous secretion signal in yeast. A large amount
of hirudin-related material detected in the culture supernatant using Western analysis is
heterogeneousandof higher apparent molecular weight than mature hirudin. Endoglycosidase
F (Endo F) treatment reduces the heterogeneity and generates a predominant form of about 15
kDa which would correspond to pro-rHV2Lys”. In the fusion protein only the MFa1 derived
‘pro’ peptide can be N-glycosylated. After amplification of the yeast KEX2 gene, which encodes
the processing protease, the amount of precursor material was diminished and the level of
biologically active rHV2Lys” was augmented more than five-fold. Mutations have been
introduced in the MFal ‘pro’ peptide, which eliminate one or more of the glycosylationsites.
All of these modifications result in significantly reduced secretion of active hirudin.
Amplification of KEX2 leads to an increase in production rates for most of the precursors
containing variant ‘pro’ peptides as comparedto the wild type form
Micro-Scale Analysis of N-Acetylneuraminic Acid
No full textWe have developed a micro-scale analytical method for the detection and quantification of Nacetylneuraminic
acid (NANA)that does neither require any derivatization of NANAnoris it interfered
by the presence of any monosaccharide component present on N-linked and O-linked carbohydrate
chains of glycoproteins. The method involves acid hydrolysis of sub-nanomolar amounts of
sialoglycoprotein (SGP) and subsequent neutralization, followed by high-pH anion-exchange
chromatography (HPAE) with pulsed amperometric detection (PAD). The method wasvalidated, using
a,-acid glycoprotein (AGP) (orosomucoid) as standard SGP. Six experimental series of decaplicates,
each, were performed, varying i) the AGP concentration in PBS (i. e. 250, 500 and 750 ug/ml,
respectively), ii) the sample amount used (i. e. 200, 100, 30, 15 and 10 ul, each, of defined AGP
concentrations), iii) the mode of sample injection into the Dionex Bio-LC system (i. e. manual or
automated injection by way of two different autoinjectors), iv) the sensitivity of the PAD detector(i. e.
1000 and 300 nAfull scale, respectively). Individual experimental series were repeated at different days
and were occasionally performed by different persons. In each case, the NANA/AGP molar ratios were
in the range of 15.0 +/- 0.4 mol/mol (corresponding with a standard deviation of <+/- 3 %), which
is in excellent agreement with the NANA/AGP molar ratio described in theliterature