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MXYOBACTERIAL POLYSACCHARIDES
No full textSpecific micromethods have been applied to the analyses of polysaccharides
produced by various strains of Myxobacteria. The polysaccharides studjed
were - associated with bacillary forms in liquid and on solid culture;
isolated from fruiting bodies; and phenol-extractable lipopolysaccharides.
A study of four Myxococcus strains indicated that polysaccharides secreted
by bacillary forms grown on solid or liquid media closely resembled
the polymers isolated from the corresponding fruiting bodies. All three
polysaccharide preparations from each strain were essentially similar
in gross composition, but differences between the strains were observed.
D-glucose and D-mannose were the major monosaccharides present in all
the polysaccharide preparations. D-galactose was present in the polymers
from three strains and N-acety1-D-glucosamine in one only. The non-identity
of polymers from different strains was confirmed by periodate-oxidation,
which revealed differential destruction of monosaccharides. It
also indicated that the polysaccharides from any one strain produced on
solid or in liquid culture and in fruiting bodies were very similar, if
not identical.
Some of the monomers present in exopolysaccharides were also found in
the lipopolysaccharides, but other monosaccharides were also detected.
Periodate oxidation destroyed all the neutral sugars present in the lipopolysaccharides.
The microcysts isolated from fruiting bodies differed
from bacilli in their polysaccharide composition; LPS was apparently
absent. In one strain, the major neutral monosaccharides detected in
hydrolysates of whole cysts were glucose and an unidentified component.
Rhamnose, mannose and galactose, the major products identified in LPS
hydrolysates, were all absent. This provides further evidence for the
absence of LPS from cysts.
These studies have been extended to other strains of fruiting Myxobacteria.
They indicate that the exopolysaccharides are composed of a very
limited range of sugars, but that differences in composition or structure
do exist between strains although probably not between fruiting
body and bacillary products. A comparision has been made between Myxozcoccus
and one Cystobacter strain in all available respects, using
bacilli and microcysts. These and other results will be discussed
IDENTIFICATION OF FERMENTATIONS BY POLYNOMIAL METHODS
No full textThe measure of the state variables of a fermentation
(biomass and substrate concentration) is always liable
to error. It is possible to reconstitute well enough
the rates of their variations during a transient state,
by a polynomial smoothing. By calculation of the derivative
polynomial, the values of the growth rate HM and
the global conversion rate R. at each moment can be deduced.
The fermentation parameters are then identified
through 2 linear smoothings.
The method is tested with data obtained by simulation
and given purposely noisy (known parameters). It is
applied in order to determine the growth parameters
of a continuous fermentation.
The advantage of this direct method upon the usual
sequential techniques is its rapidity and its small
occupation of the central memory of the computer.
It can be applied to continuous or discontinuous fermentation
MÖGLICHKEITEN DER ENZYMATISCHEN SAUERSTOFFENTFERNUNG UND KONSERVIERUNG IM LEBENSMITTELBEREICH
No full textZusammenfassung: In jüngster Zeit ist ein verstärktes Interesse am Einsatz von Enzymen als biologische Alternativen zu zwei wichtigen Gruppen
von Lebensmittelzusatzstoffen zu verzeichnen, Antioxidantien und Konservierungsmittel. Am Beispiel von Glucose-Oxidase als Sauerstoffänger
werden Vor- und Nachteile derartiger Systeme aufgezeigt und Grenzen aus
technologischer-, wirtschaftlicher- und rechtlicher Sicht angesprochen.
Ausgehend von in der Natur entwickelten Strategien zur Verhinderung
mikrobiellen Befalls lassen sich ebenfalls Konzepte für enzymatische/-
biochemische Konservierungsmittel entwickeln. Im speziellen wird eingegangen auf Glucose-Oxidase, Lactoperoxidase/Lactoferrin, Lysozym und
Nisin.
Abstract: Recently enzymes have been recognized as possible natural substitutes for two important classes of food additives: antioxidants and
preservatives. With glucose oxidase as an example for a biological
oxigen scavenger potential and limits of such alternative systems from a
technological, economic as well as legal point of view will be
discussed. Based on nature's strategies to present microbial attack
concepts for enzymatic and/or biochemical food preservations can be
developed
AKTUELLE BIOCHEMISCHE METHODEN IN DER LEBENSMITTELANALYTIK
No full textAn overview is given on the use of methods, mainly developed in
biochemistry, in the field of food analysis. Special emphasis is
given to enzymatic assays as well as to enzymes in milk. Beside
this, the usefulness of antibodies in food analysis is demonstrated.
Last not least, typical biochemical methods of separation techniques
like column chromatography of macromolecules and separation of
proteins in electric fields, especially on ultrathin layers, are
considered
COMPUTERAIDED PROTEIN DESIGN: METHODS AND APPLICATIONS
No full textSince the first reports on the use ofsite directed mutagenesis in 1982! protein engineering or - when
rationally aimed - protein design has been Tecognized as a promising and fascinating field of
research in many countries. In Japan (PERI) and the U.S.A. (CARB)researchinstitutes have been
founded with the focus on protein design. More and more researchinstitutes in the United States,
Canada, Japan and Europe have been Starting broad research projects on protein design (UK:
SERC, W. Germany: GBF, EMBL).
Possible prospects for applications of designed proteins with modified activities or other new
properties are very high,in the areas of pharmacology, enzyme applications in food industry’, waste
treatment and chemical synthesis, vaccine design, biosensors etc.*". This conception wasvery clearly
lined out in an excellent article by Kevin Ulmerin 19835,
Encouraging results have so far been obtained only for a small number ofcases including insulin,
proteases and peptidic protease inhibitors, and some others®’. On the other hand many unpredicted
andsurprising results of site directed mutagenesis experiments are reported onscientific meetings
and in the literature*’, That demonstrates that our methods andtools in thatarea arestill rather
crude and urgently require improvement!",
Simplecalculations show that the random approach to protein-engineeringis a very slow one. There
are 10°* ways to arrange aminoacids in a medium sized protein chain of 250 amino acids. Ca. 10”
molecules would form the whole estimated mass ofour universe. But even whenthe information
about the seven most important aminoacids is available and only five changes should be tested
for each of the positions, about 80,000 different protein-mutants have to be prepared andtested.
This implies that a knowledge of the 3D-protein-structure and a good understandingofthe functionactivity
relationship is absolutely essential in order to do rational protein-design.
Research projects in protein-design require a close cooperation between groupsspecialising in
protein-isolation and purification, in fermentation techniques, in genetic-engineering, in DNAsynthesis
and protein-crystallography (protein-NMR techniques are being established). This
interdisciplinary connection between protein chemists, molecular biologists and stereo-chemists is
essential for the protein-design cycle (Fig. 1) consisting of design, cloning, expression andtesting
new proteins starting from known ones
DESIGN AND STRUCTURES OF DISULFIDE CONTAINING SUBTILISIN VARIANTS
No full textThe crystal structures of 4 variants of subtilisin, each one containing an engineered
disulfide crosslink have been determined. The geometries of the engineered disulfide
groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a
relationship between their measured redox potentials and their calculated dihedral
energies. Disulfide introduction produced cavities in the protein structures. The cavity
produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was
partially filled by a disordering of nearby Asn117. The cavities were often filled with
ordered water molecules that replaced interactions of the removed groups. Molecular
modelling provided insight into the location where a disulfide could be incorporated, and
into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed
that introduction of disulfides into buried hydrophobic regions resulted in long range
concerted rearrangements
STRUCTURAL AND FUNCTIONAL ASPECTS OF PROTEIN-PROTEIN INTERACTION AS STUDIED THROUGH CRYSTAL STRUCTURE OF SUBTILISIN COMPLEXED WITH ITS TRAPPED SUBSTRATE SSI (STREPTOMYCES SUBTILISIN INHIBITOR)
No full textThe crystal structure of Streptomyces Subtilisin
Inhibitor (SSI) was partially refined by restrained least-squares
methods to a conventional R value of 24 % employing rotating
anode data to 1.85 A resolution range. The Sstructines of the
complex of a bacterial alkaline serine proteinase, subtilisin
BPN’, with its proteinaceous inhibitor SSI was partially refined
to&® the (sR valuesgot 216% seupkoying lag RR synchrotron data.
Comparing the B-factors between free SSI and complexed SSI, the
marked rigidification of polypeptide chain segments occurred not
only in the “reactive site segment” which is in direct contact
with the enzyme but also in those segments which are closely
connected with the reactive site segment through either covalent
linkage or non-covalent interactions. Moreover the structure of
the complex of subtilisin with genetically engineered mutant SSI
was solved by ( F mutant - F wild ) difference Fourier syntheses
Podiumsdiskussion
No full textDie Diskussion bewegte sich, nach einer kurzen Vorstellung der gezeigten Poster zu Beginn, im
wesentlichen um folgende Themenkreise:
- gesetzliche Rahmenbedingungen für den Einsatz von Enzymen
- Forschungspotential und wirtschaftliche Risiken der mittelständischen Industrie
- zu erwartende analytische Anforderungen nach der Schaffung des europäischen Binnenmarktes.
Die nachstehende Widergabe der Diskussion folgt dem zeitlichen Ablauf
NOVEL BIOSENSOR SYSTEMS FOR CLINICAL AND FOOD ANALYSIS USING MICRO DEVICES
No full textThe amorphous silicon ion sensitive field effect transistor (a-ISFET)
made by radio frequency plasma discharge was used as a pH-sensitive
device. The size of the channel of the a-ISFET is 10 um long and 500
um wide. The pH sensitive layer was silicon oxide evaporated over the
amorphous silicon nitride layer. The pH sensitivity was about 46
mV/pH at 18°C in the range pH 5-10. The response times of this device
to pH change are very rapid, being less than 30 sec to reach a steady
state value. The fish freshness sensor was constructed by using an
immobilized xantine oxidase membrane and a-ISFET. Hypoxantine was
detected in the range 0.02-0.1mM. Micro-biosensors for glucose and
glutamate were constructed by using silicon fabrication technology. A
linear relationship was observed between the glucose concentration in
the range from 0.1 to 10 mg aı-!, and the decrease in current.
Glutamate was determined in the range from 5 to 50 mM. The microoxygen
electrode consists of two gold electrodes and agarose gel
containing electrolyte covered with a gas permeable membrane. A
negative photoresist was used as the gas permeable membrane which was
directly cast onto the gel and submitted to photochemical reaction.
A 90% response time of the micro-oxygen electrode took approximately 3
minutes. A linear relationship was obtained between the response of
the micro-oxygen electrode and that of a conventional Galvanic oxygen
electrode
RECENT DEVELOPMENTS OF BIOSENSORS BASED ON THERMISTORS AND SEMICONDUCTORS
No full textThe enzyme thermistor (ET) is a simple flow calorimeter primarily intended
for rapid metabolite assays with use of immobilized enzymes,
but it can be used as a general biocalorimeter as well. The reaction
heat produced in a small column containing immobilized biocatalyst is
measured with a thermistor as a temperature change of the effluent of
the column. The temperature change is linearjvs. the substrate) icon—
centration over wide ranges from about 1 uM to several hundred mM depending
on the actual enzyme reaction. Considerably higher sensitivities
can be obtained by using recycling enzyme systems. Up to 60 samples
per hour can be analyzed. A large number of metabolite assays of
clinical as well as of biotechnological interest has been studied.
The ET assay can easily be automated and is well suited for monitoring
and control of biotechnological processes due to its high operational
stability. For the monitoring of larger molecules a thermometric enzyme
immunoassay (TELISA) has been developed and automated.
Hydrogen- and ammonia-sensitive semiconductors of Pd-MOS type could be
combined with enzymes and cells into highly sensitive biosensors.
Addition of another catalytic metal, such as Ir on the gate of the MOSdevice
results in enhanced ammonia-sensitivity. Highly sensitive methods
for the determination of e.g. urea and creatinine have been developed.
Detection is made in the gas phase, which is advantageous