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ENZYMATIC MULTI-CHANNEL-FIA METHODS FOR ONLINE FERMENTATION MONITORING AND CONTROL
No full textA multichannel FIA system with high reliability and flexibility was developed
for on-line process monitoring and control of fed batch and continuous fermentations.
Glucose, L-Lactate, D-a and L-a-Hydroxybutyric acid(D- und LHBS),
Isoleucin, Leucin, Ethanol, Acetaldehyde and Ammonia are enzymatically
determined with fluorimetric detection. For all assays dehydrogenases alone
or coupled with a second enzyme are immobilized in packed bed tube reactors.
In the proposed parallel configuration of enzyme reactors only one detector
is necessary. In a miniaturized and modular on-line sampling system
the sample solution is diluted, conditioned and purified from interfering
substances. The proposed FIA set up allows the adaptation of the different
analyte concentrations to the ranges of the corresponding enzymatic assays.
A freely programmable valve configuration guarantees a high flexibility in
solving different problems of process analysis. The whole FIA-equipmentis
controlled by an user friendly software running on a personal computer.The
process analytical regime consists of on-line analysis, calibration- and
recalibration cycles. The control of these cycles, the detctor signal
evaluation and the calculation of the analytical results are implemented fully
automatic
STRUCTURAL STUDIES OF GEOTRICHUM CANDIDUM LIPASE
No full textLipase from Geotrichum candidum has been purified to homogeneity and
crystallized in three different forms. Rotation function analysis indicates close
relationship in molecular packing in all forms. Despite intensive search only one
isomorphous heavy atom derivative has been found to date. A further search is in
progress
CRYSTALLOGRAPHIC STUDY OF A RECOMBINANT CUTINASE FROM FUSARIUM SOLANIPISI
No full textCutinases are a group of extracellular fungal hydrolytic enzymes capable
of degrading the insoluble lipid polyester matrix,i.e. cutin, which covers the
surface of plants. 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 yieldsin E. coli cultures. Cutinase wascrystallized (PEG 6000 15-20% ,pH 7.0
to 10.0,20°C) in space group P21 with cell dimensions 35.1A,67.4A,37.05 A,
ß=94°.They diffract to 1.5 A resolution (Rsym=4.41%). Data from native and derivatives
have been collected. MIR phasingis in progress
Human Lipoprotein Lipase: Important Roles Of A Specific N-Linked Glycosylation Site And Specific Serines In Secretion And Enzyme Activity
No full textThestructure-function relationship of human lipoprotein lipase was studied by expressionof the
cloned cDNAin transfected cells, using the wildtype construct and site-specific mutant constructs.
Asparagine 43, of one ofthe two potential N-linked glycosylationsites in lipoprotein lipase, was found
to be important for both enzymeactivity and secretion. Mutations involving someoftheserine residues
also produced marked changes in enzymeactivity. The possible structural changes associated with
these functionally altered mutantlipoprotein lipase molecules are discussed with reference to the crystal
structure of human pancreatic lipase, a lipolytic enzyme with considerable sequence homology to
lipoprotein lipase
Physicochemical Properties of Mono- and Diacylglycerol Lipase from Penicillium camembertii
No full textIn the course of investigations on the enzymatic synthesis of monoglycerides and the partial
hydrolysis of glycerides, Yamaguchi and Mase found a newlipase havingstrict specificity to
mono- and diacylglycerols but not to triacylglycerols in the culture broth of Penicillium
camembertii U-150 (1). The newlipase was purified into four active fractions by a procedure
involving ethanol precipitation, ammanium sulfate fractionation, and aminooctyl-Sepharose,
hydroxyapatite and concanavalin A-Sepharose (con A Sepharose) column chromatographies.
One active fraction, enzyme 1, was not adsorbed on con A-Sepharose but others, enzymes 2-
4, were adsorbed on con A Sepharose and separated into three active fractions by linear
gradient elution with Methyl-X-D-glycopyranoside.
No significant difference was observed in substrate specificity among enzymes 1-4, but other
enzymatic properties, e. g., pH and heatstabilities, and optimum pH and temperature, were
clearly different between enzyme 1 and three adsorbed components (three adsorbed
components weresimilar to each other)(2).
In oder to elusidate multiple forms of this enzyme, the physicochemical properties were
compared among four active components
LIPOLYTIC ENZYMES SEPARATION AND PURIFICATION THROUGH FUNCTIONALIZED SYNTHETIC POLYMERS
No full textA new class of functionalized synthetic polymers was prepared for
the purification of lipases by a single step affinity chromatography.
Polyvinyl alcohol polymers were crosslinked with epichlorohydrin and
esterified with fatty acids of different length. The resulting resins
were characterized by infrared spectroscopy, electron microscopy and
titration of the hydrolysed product to evaluate the degree of
esterification. The attention was focused on lauryl ester of polyvinyl
alcohol as commercial preparation of Candida cylindracea lipase showed
the highest enzyme affinity for esters of fatty acids with a linear
chain ranging from 8 to 12 carbon atoms. Chromatographic lipase
purification trials were performed on a 7 cm x 1.6 cm i.d. column.
Good separation conditions were found by utilizing stepwise increase
in CHAPS (3-(3-(cholamido-propyl)-dimetil-ammonio)-1-propan sulfonate)
concentration in HEPS/EDTA buffer. The lipase obtained by elution with
CHAPS 6.0 mmol/L showed a high purity, as established by SDS-PAGE,
where an unique band with a molecular weight of 60.000 was identified
USE OF BIOSENSORS IN ANALYZERS FOR CLINICAL-CHEMICAL LABORATORIES
No full textPrüfgeräte-Werk Medingen GmbH is a middle class enterprise resident
in the Dresden-Freital region. Since 1968, it has been manufacturing
analyzers using the flow stream principle.
Experience gathered during that period brought about in cooperation
with Zentralinstitut für Molekularbiologie Berlin the application of
the flow stream principle aiso for measurements using biosensors. A
result are micro flow cells for determination of glucose or lactate in
blood, serum or plasma which nowadays are being used in about 1.000
analyzers of the types ECA 20 and ESAT 6660.
The measurement is based on detection of H202 being produced by the
known reactions
GOD
8-D-glucose + 02 + H20 —™ D-gluconic acid + H202
LOD
L-lactäte#403%+ 238230) —e pyruvateii+'H303.
Thus, a signal is obtained that is independent of the oxygen
content in the blood.
Easy handling, an optimized automatic regime of measurement and the
availability of preconfectionated membranes and solutions are some
reasons for the broad application
MEMBRANE MATERIALS FOR AMPEROMETRIC AND POTENTIOMETRIC THICKFILM BIOSENSORS
No full textThickfilm transducers are reliable devices for the construction of biosensors. Polymer mixtures
are described which can be used in screen printing processes and allow to immobilize biopolymers,e.g.,
enzymes, effectively. Potentiometric membranes with improved adhesionto the thickfilm electrodes are
presented. Examples from the field of potentiometric (urea, penicillin) and amperometric (glucose,
sucrose, ascorbic acid) enzyme sensors are reported and compared to conventional electrodes. The
application of a 4-channel multisensor for the determination of glucose, sucrose and ascorbic acid is
described
NEW DEVELOPMENTS IN THE FIELD OF BIOSENSORS- APPLIED TO THE DETERMINATION OF PESTICIDES IN WATER
No full textThe application of new methods and biological components to the determination of pesticides in
water is described. Triazines were monitored by competitive immunochemical assays applied to
flow-through systems. With a FILA, which was based onthe principle of an ELISA,
determination of pesticides within the limits of the European drinking water act was possible
without preconcentration of the sample. Alternatively, a fluorescence-labelled antibody was used
andthe level of binding measured using the method of the evanescent wave technique. With this
system a regeneration was possible up to 300 times without anyloss ofactvity.
The determination of particular pesticides was supplemented by the development of biosensors
for a class of pesticides. This was achieved by microbial sensors for chlorinated compounds and
by a test based on the inhibition of cholinesterase by carbamates and organophosphates. The
microbial cells of Alcaligenes eutrophus JMP 134 were immobilized on an oxygen electrode, an
increase of oxygen uptake being observed in the presence of 2, 4-dichlorophenoxyacetic acid and
its derivatives. The inhibition of cholinesterase was monitored either automatically by a flow
injection system or by a disposable sensor madebythick film technology
NICKELOCENE AS ELECTROCHEMICAL MEDIATOR FOR GLUCOSE OXIDASE IMMOBILIZED ON PYROLYTIC GRAPHITE ELECTRODE
No full textNickelocene is investigated as an electrochemical
mediator in glucose oxidase amperometric electrodes. The
formal redox potential of nicKelocene /nickelocenium obtained
in adsorbed state on electrochemically pre-treated (activated)
pyrolytic graphite electrode,is -0.1 V (vs. Ag/AgCl). Adsorbed
nickelocene mediates the charge transfer between the electrode
and glucose oxidase. The steady-state amperometric response
of nickelocene mediated enzyme electrode is investigated as a
function of the glucose concentration, applied potential and
pH of electrolyte