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MEDIA ENGINEERING IN THE CATALYSIS BY LIPASES
No full textTwo reactions catalyzed by lipases have been studied, namely i)
Transesterification between ethyl butyrate and glycerol in a two-phase
system. The optimal water content in the mixture was 5%. Only
monobutyrin was obtained. ii) Hydrolysis of p-nitrophenyl esters in
microemulsions of sodium bis-2-ethylhexyl in heptane; its specificity
constant was an order of magnitude lower than in aqueous mediun
PROPERTIES AND PARTIAL PURIFICATION OF A PSEUDOMONAS CEPACIA LIPASE
No full textA high lipolytic activity (65 umol/ml min.) was observed in the supernatant of
Pseudomonas cepacia DSM 50181 after growth on olive oil. The crude lipase was
investigated with respect to substrate specifity, pH- and temperature optima. The
hydrolysis of short chain triglycerides and of the unsaturated triolein indicated a
higher lipolytic activity than in the case of long chain substances (37°C). The p-nitrophenyl-
esters of fatty acids showed maximum activity for the palmitate. This was
the same maximum which was observed for triglycerides in the range from C12 to
C18 at 75°C. An inhibition of lipolytic activity was noticed by adding oleic acid
during the hydrolysis of olive oil. Furthermore an inactivation of the lipase occured
when the interface liquid/gaseous was increased. The addition of an inert lipophilic
substance or of detergents could neutralize this effect. A partial purification of the
lipolytic enzyme could be achieved by liquid/liquid extraction and ionexchangechromatography.
This enrichment procedures led to a purification factor of 55 with
a recovery of 30%. Isoelectric focussing showed a main protein band at pl 7.1
Comparison of Lipase Activities by Different Assays
No full textRecently several quick assay procedures have been developed (1 to 15), and are saidto detect
lipase activity. The hydrolysis of Triglycerols and artificial substrates oftencan not explain
differences of whatis a lipase and whatis an esterase. We established a numberoflipase assays
for the estimation ofactivities for a large numberof different lipase preparations, as immobilized
pure enzymes, nearly homogeneous preparations and samples of industrial grade. We found
remarkable differences concerning the lipase activities measured with different assays and report
about characteristics and comparability. At last, we want to arise the question "do we look for
the enzymes we are interested in ?
GLUCOSE OXIDASE/LIPID MIXED LB-FILMS ON A PT ELECTRODE APPLICATION AS SENSOR MODEL
No full textGlucose oxidase (GOD)/lipid Langmuir-films on the air/water
interface were prepared and investigated. The enzyme molecules
were adsorbed by a preformed pure or mixed lipid monolayer from
the aqueous subphase of the Langmuir-trough. The following types
of lipids and their mixtures were used: lecithine, lecithine/
cetyltrimethylammonium bromide (CTAB), CTAB-modified methylmethacrylate
monomer, stearic acid and stearic acid/CTAB. GOD was
found to adsorb the strongest on positively charged lipid monolayers.
It is due to the negative charges dominating the protein
molecule at pH 7.0. The enzyme retains its enzymatic activity in
adsorbed layers. The transfer of one to ten lipid-protein monolayers
onto Pt-electrodes under 20-40 mN/m surface pressure was
performed by the Langmuir-Blodgett method. As a result the biosensor
was obtained with a measuring range from 0.5-5.0 mM
glucose. The current output was found to depend on the number of
transferred lipid-protein monolayers and on the surface pressure
at which the transfer was carried out. The best electrode
characteristics were found with glucose oxidase adsorbed on monolayers
of the CTAB-modified methylmethacrylate monomer
BACTERIORHODOPSIN VARIANTS FOR OPTICAL INFORMATION PROCESSING
No full textBacteriorhodopsin (BR)is a biological photochromecontained in the purple membrane (PM) from
Halobacterium halobium. BR-films can be used as reversible recording materials for holography.
Mutagenesis of the halobacterial gene coding for the bacterio-opsin protein is the key to generate
BR-variants with modified optical properties. BR-films with improved holographic characteristics, i.e.
increased diffraction efficiency and sensitivity, containing mutated bacteriorhodopsins have been used in
dynamic holographic recording, real time interferometry and holographic pattern recognition
BIOSENSORS BASED ON RECEPTORS : IMMUNOSENSORS ON THE BASIS OF LIPOPEPTIDES
No full textCovalent attachment of the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-
[2RS]-cysteine (Pam3Cys-OH) (10) - a synthetic analogue to the N-terminal part of lipoprotein
from the outer membrane of E. coli (11) - to peptidic antigens (epitopes) results in lipopeptideconjugates
with interesting immunological and biophysical properties. Lipopeptides are able to
activate B-lymphocytes (10,12), macrophages (13) and neutrophilic cells (14) and induce the production
of specific antibodies against the antigenic peptide (15). The developmentof a fully synthetic
vaccine against foot-and-mouth disease (FMDV) (16) as well as the in vivo-priming of
cytotoxic T-lymphocytes (17) demonstrate the immunological power of lipopeptides. Biophysical
investigations with labelled lipopeptides (18-20) show that lipopeptides have a high tendency to
insert into membranes of living cells and into artificial phospholipid membranes (vesicles). The
high affinity of lipopeptides to hydrophobic surfaces has been used for the coating of polystyrene
microtiter plates in an assay (ELISA) for the distinction between HIV-1 and HIV-2 (21)
DIRECT OBSERVATION OF ANTI-ATRAZINE ANTIBODY BINDING USING GRATING COUPLERS
No full textPlanar monomode waveguides with grating couplers have beenapplied for the direct detection of
anti-atrazine antibodies and a pesticide assay has been developed on the basis of a competitive
immunoassay. Data are shown for the binding of anti-atrazine antibodiesin the concentration range
from 1 to 10 ug/ml to an immobilized atrazine-derivative, corresponding to a changein the adlayer
thickness of between 0.1 and 3 A. First competition experiments using terbutryn showed significant
suppression of antibody binding at a concentration of 10 ng/ml
SIMULTANEOUS FLOW INJECTION ANALYSIS OF L-LACTATE AND L-MALATE IN WINE BASED ON THE USE OF ENZYMES
No full textThe simultaneous determination of L-malate and L-lactate, by enzymesupported
FIA, was developed using two enzymereactors in parallel and a
single oxygen electrode. NADH formed in the reaction of malate dehydrogenase
(MDH) was regenerated to NAD with dissolved oxygen,using vitamin K3
and diaphorase (DI). L-Lactate was determined using the enzymelactate
oxidase (LOD). When sample solutions were simultaneously injected into the
two reactors (the MDH-Di-reactor and the LOD-reactor) with a controlled
residence time, a train of two peaks corresponding to L-lactate and L-malate
were seen in the FIA-gram. The peak currents were linearly related to the Lmalate
and L-lactate concentration in the range 0.05-1.2 mM and 0.01-0.5 mM,
respectively. The present system was applied to the determination of Llactate
and L-malate in white wine. The results showed a good agreement with those
obtained using a conventional method (F-Kit method), suggesting that this
system may be applicable to the monitoring of malo-lactic fermentation during
wine production
CHARACTERISTICS AND APPLICATION OF A CONTINUOUS GLUCOSE REGISTRATION DEVICE USING THE MICRODIALYSIS TECHNIQUE
No full textA device for continuous registration of dissolved glucose was obtained
by combining the microdialysis technique with the measuring flow chamber
of a glucose monitor using the glucose oxidase method for the determination
of glucose concentration. In in vitro experiments it was
demonstrated that the glucose signal registered by this device is inversely
related to the flow rate, which, in turn, is inversely related
to the response time of the device. The glucose signal increased linearly
with the area of the microdialysis working membrane and with the
glucose concentrations of the standard solutions. The influences of
temperature changes of the glucose standard solutions upon the glucose
signal were determined by 1.32%/degc. In in vivo experiments, a needle
type microdialysis probe was implanted subcutaneously in rats, whose
blood glucose concentration was varied between 40 and 122 mg/dl. Blood
glucose concentration was followed closely by the glucose signal obtained
from the subcutaneous probe (r = 0.94) over a 240 min registration
time period
IMPORTANCE OF HETEROGENEOUS ELECTRON TRANSFER IN MONOENZYMATIC AND BIENZYMATIC ELECTROCHEMICAL SENSORS
No full textAbout several examples developed in the laboratory we
discuss of the role of electronic transfer between electrode and
ions or biomolecules in the increase of sensitivity and
selectivity of biosensors.
According to the kind of enzymes used in these biosensors it was
possible to propose:
- a monoenzymatic L.lactate electrode based on the direct
electron transfer between platinum and a lactate dehydrogenase
containing flavines and hemic groups;
- bienzymatic electrodes for L.lactate, D.lactate and L.carnitine
using both dehydrogenase and diaphorase and exploiting the
principle of enzymatic amplification;
- electrode for NADH using a double mediation with formate
dehydrogenase and flavins