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Bacterial Luciferase of Vibrio harveyi MAV: Purification, Characterization, and Application
No full textIn the medical and biotechnological industry is an increasing interest for bioluminescent systems for
example as analytical tools and genetic markers. We introduced a new procedurefora large scale purification of
luciferase from Vibrio harveyi MAV in view of subsequentcrystallization of the enzyme. In four steps the luciferase was
obtained in greater than 99 % purity using the chromatography equipment (FPLC-System)from Pharmacia/LKB, Freiburg,
Germany. Homogeneity of the luciferase was proved by SDS-PAGE andsilver staining. The enzyme was
purified 34fold to a yield of 29 % and a specific activity of 1.8*10'' [LU/mg]. The optimum activity of luciferase was
detected at pH 6.8 and 30°C.Theisoelectric point proved to be at pH 4.7. Treatment with the inhibitors diethylcarbonate,
phenylmethylsulfonyifluoride, and diethyl-p-nitrophenylphosphate until unknownasluciferase inhibitors, affected
the activity with the same extent and nearly the samevelocity as other inhibitors, reacting with histidine and cysteine
residues at the active center of luciferase. The enzymecrystallized depending upondifferent precipitation agentsin
various forms. We obtained twotypesof crystals, needles in ammonium sulfite and rhombic-like in ammonium sulfate
THE USE OF REDOX MEDIATORS FOR AMPEROMETRIC BIOSENSORS
No full textThe paper describes redox mediators enabling an efficient electron
transfer between biological redox systems and the electrode surface in
amperometric biosensors. Criteria for the selection of appropriate
mediators are discussed. Different technological aspects of anchoring
the mediator by a simple physical entrapment into the electrode
material of both conventional electrodes and screen-printed forms are
illustrated. Investigations are performed with tetrathiafulvalene and
several quinoid redox-dye derivatives serving as redox couples for
glucose oxidase, lactate oxidase, peroxidase, "phenol oxidase",
bacteria, NADH, and cytochrome c. Characteristics of several enzyme
electrodes are presented
RAPID MICROBIAL TESTING IN ‘REAL’ SAMPLES
No full textThree examples of electrochemical approachesto rapid assessmentof the microbiological
Status of materials, especially foodstuffs, are described. One concerns the monitoring of overall
reductive capacity for the determination of total microbial numbers in foodstuffs. A production
prototype has now been developedfor the dairy industry. Another approachinvolvesthe useof
a multiple, mediated amperometric glucose sensor to determine the glucose profile
perpendicular to the surface of uncooked meat. This is affected by the surface microflora and
hencethe principle can be used asthe basis of a meat freshness sensor. The final exampleis
based on the exploitation of electrochemical immunoassay for identification of specific
microbial markers
BIOSENSOR SYSTEMSUTILIZING NADH AS AN INTERMEDIATE
No full textThe combination of dehydrogenases and chemically modified graphite electrodes has proved
particularly useful in the developmentof highly selective and sensitive biosensor systems.
Amongthe applications given is a flow injection system for the determination of fructose
with a linear range of 1 pM - 2 mM.As a means toincrease sensitivity further, substrate
recycling was employed in the determination of ATP down to the nano-molar level. A
creatine and creatinine assayillustrates the potential of coupled equilibria and coimmobilized
enzymes. For the determination ofglucose in whole blood a dialyzer was integrated with a
biosensor. The hematocrit dependence on the transfer of glucose through the dialysis
membrane was studied
Statistical Analysis of DNA Sequences
No full textWereport recent results of statistical analysis of DNA sequencesin this paper. First,
we show howrepetitive segments reduce the information content measured by Shannon
entropies. Then, the effect of a nonuniform codon usage on the mutual information
function is studied analytically. For this purpose, the concept of pseudo-exons is introduced.
Finally, we discuss the modularity of DNA which can serve as a sensitive tool to
distinguish DNA sequences from randomstrings
GLYCOSYLATION OF THE INFLUENZA VIRUS HEMAGGLUTININ IN INSECT CELLS
No full textThe hemagglutinin of influenza virus grown in chick embryo cells has N-glycosidic carbohydrate
side chains which are of the complex type at most and of the oligomannosidic type at a few attachmentsites.
When the hemagglutinin was expressed in insect cells using a baculovirus vector, it was
found that these cells have the capacity to trim N-glycans to ManzGlcNAc, cores and to further
process these by the addition of fucose to Manz[Fuc]GleNAc,. Thus, the complex oligosaccharides
found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect
cells by small truncated side chains. The effects of the truncated oligosaccharides on maturation of
the hemagglutinin and onits biological functions will be discussed
PHOSPHOLIPID-GLYCERIDE INTERACTIONS AS REGULATORS OF CARBOXYLESTER LIPASE ADSORPTION AND CATALYSIS
No full textLipolysis occurs in the presence of non-substrate, surface-active
molecules like phosphatidylcholines (PC’s). The role of PC in the
regulation of carboxylester lipase (CEL) was investigated using mixed
lipid monolayers. For each substrate an abrupt increase in substrate
hydrolysis from 90%, i.e. switching, occurred at a substratedependent
lipid composition. Similar results were obtained for free
fatty acid 180 exchange catalyzed by CEL. The lack of hydrolysis at
low substrate was not due to an absence of CEL at the interface.
Enzyme adsorption to substrate.PC films is consistent with all surface
being available, except for 43.5 AZ /molecule of PC. Adsorption of CEL
obtained by extrapolation to infinite dilution of PC was 0.92
pmol/cm?, 1/4 of the expected value. CEL adsorption to PCefatty acid
films fell short of the values predicted from substrate-containing
films. These results suggest that adsorption of CEL to PC-rich films
is regulated by clusters or domains of substrate or fatty acid
molecules in the PC interface. Such clusters are predicted from
stochastic distribution of components but appear to be modulated by
lipid-lipid interactions. The stochastic argument further predicts
that substrate domains should interconnect if >45% of total surface
area is non-excluded by PC. Using data obtained from CEL binding to
calculate non-excluded area at each lipid composition, hydrolysis and
180-exchange data are consistent with catalysis being regulated by
domain connectivity, i.e. percolation. Being lipid-based, this
regulation should occur with other lipases, but the consequences of
the organization may be lipase specific
PRODUCTION AND SOME PROPERTIES OF PARTIALLY PURIFIED LIPASE FROM PENICILLIUM SIMPLICISSIMUM
No full textLipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are widely
distributed throughout fungi. Two lipolytic fungi commonly used in the
manufacture of Camembert and Brie cheeses are Penicillium camembertii and
P. caseicolum. Lipolytic activity was also discovered in a P. chrysogenum
strain [1]. Lipase from P. cyclopium M1 specific to p-nitrophenyl laurate
has been purified and crystallized [2]. Three lipases were discovered in
the culture broth of P. crutosum [3], two of them were purified and
crystallized [3]. We describe here a lipolytic activity of P.
simplicissimum, the purification and some properties of the lipase
BIOSENSORS FOR CONTINUOUS EX-VIVO DETERMINATION IN CONJUNCTION WITH AN ARTIFICIAL PANCREAS
No full textGlucose, lactate, and pyruvate can be continuously measured in whole blood by
using three extracorporeal electrochemical biosensors.
We fixed two newly developed sensors, for lactate and pyruvate, in series with
an endocrine artificial pancreas ("Betalike") without affecting its performance.
The new sensors for whole blood should be able to clarify the fate of the
intermediary metabolites of glucose in diabetic patients when drugs or insulin
is infused
DISTRIBUTION OF CELLULOSE DEGRADING GRAM-NEGATIVE BACTERIA
No full textNew methods have been developed for the enrichment, purification and characterization
of cellulose degrading bacteria from brackish, freshwater and terrestrial habitats.
The methods involve the use of bacterial cellulose mats as cellulosic substrates
for rapid identification and purification of these organisms. A preliminary
survey of 53 isolates of mesophilic aerobic and facultatively anaerobic organisms
reveals the following generalizations. (1) All the organisms were Gram-negative and
no flagellates were found. (2) From any one habitat the cellulose degraders form
tight clusters of related types. (3) The brackish samples from a salt marsh produced
two novels types of myxobacteria, tentatively allocated to Polyangium. (4)
From freshwater samples, the dominant types were cytophagas (5) An interesting
group of organisms capable of degrading cellulose, agar and gelatin was discovered