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Lipase, Enzymatically Active in a Monolayer
No full textThe present work is focussed on the interfacial behaviour and the enzymatic activity of Staphylococcus
carnosus lipase monolayersat the water/air and water/tolueneinterface.
Stable enzyme layers were established at the water/air interface by quantitative spreading according to
Trurnit (1). Thus, a monolayer with a definite number of molecules is obtained, exclusively. Therefore the resulting
pressure/area diagramscanbeinvestigated with regard to molecular data.
A characteristic feature is the occuranceof a critical pressure 7, ~ 18 mN/m in the pressure/area diagrams.
This critical pressure occurs in many protein pressure/area diagrams. In the caseoflipase, Tig is pHindependent
and can be demonstrated to correspond to a closely packed monolayer of compact molecules.
From the corresponding molecular area A. (= 5000 A2) the molecules can be postulated to be discs
lying flat on the surface and the radius and thickness can be calculated to 40 Aand 20 A, respectively.
The covering of the monolayer with a toluene-phase changes the intermolecular interactions: The
pressure/area curve progresses beyondthe corresponding water/air curve. For pressureshigher than rm,
and molecular areas smaller than A, the difference diminishes. The values of m,/A, at water/air and
water/toluene were the same. Asfor the water/air interface thecritical pressure is pH-independent.
For studying enzymatic activity at the water/air interface a novel apparatus is presented, which combines a
filmbalance with a convection reactor. Thus, it is possible to study the enzymatic reaction of a definite monolayer
in a defined hydrodynamical environment. The enzymatic monolayerturns out ta be active and its
reaction resembles the corresponding homogeneous reaction. Thus support is given for the view,that
enzymes are not necessarily denatured on their contact with an interface, but can keep a compact and
active configuration
Purification and Kinetic Studies of Lipases Using Reversed Micellar Systems
No full textSelective separation and purification of two lipases (A and B) from
Chromobacterium viscosum were carried out by liquid-liquid extraction using a
reversed micellar system (250 mM AOTin isooctane), with selective extraction of
lipase B to the micellar phase and lipase A remaining in aqueous solution. The
purified lipases were used to catalyze esterification reactions between carboxylic
acids and alcohols in reversed micelles. The influence of various parameters in the
synthesis of oleyl propionate by lipases A and B, was studied. The maximum activity
was obtained at Wo([H20]/[AOT])=6.7
ENZYME ELECTRODES FOR MEDICAL APPLICATIONS
No full text1.The development and optimization of a highly sensitive creatine
sensor based on the creatine amidinohydrolase - sarcosine oxidase
sequence is described. The bi-enzyme sensor for the analysis of
creatinine and creatine kinase is characterized by the following
analytical performance: response time below 15 s, detection limit 5 uM
creatine, linear concentration range up to 500 uM creatine, functional
stability more than & days, serial imprecision below 5 %, measuring
range for CK between 1 and 1000 U/l. 2. A well designed modified lactate
sensor based on a cellulose-lactate oxidase/polycarbonate-cellulose
membrane system is demonstrated for the accurate analysis of lactate in
a concentration range between 0.2 and 20 mM using undiluted whole
blood. This sensor now is commercialized as the main part of the
BIOSENLactat2000 (EKF Industrie-Elektronik Magdeburg, Germany)
HOW TO IMPROVE ENZYME ELECTRODES ?
No full textThe mathematical background of two-step amperometric enzyme electrodes is
presented
THE STRUCTURE OF LIPASE GENES AND PSEUDOGENES OF CANDIDA CYLINDRACEA
No full textCandida cylindracea produces at least two kinds of extracellular lipase,
lipase I and lipase [. From the mutant which produces a large amount of
lipase I and a negligible amount of lipase [[, we obtained the lipase I cDNAs
and genomic genes. Lipase I genes were classified into several types by
sequencing and restriction analysis. Lipase I proteins were thought to be
encoded by several homologous genes. Lipase I was homologous with Geotrichum
candidum lipases. Furthermore, we obtained pseudogenes showing more than 90%
homology with lipase I cDNA.
Although the amino acid composition of purified lipase I was consistent with
that deduced from the cDNA sequence, significant discrepancies were evident in
the contents of leucine and serine. These discrepancies are due to the
different genetic code used by C. cylindracea, in which the universal codon for
leucine, CUG, is used to code for serine
THE LIPASE GENE OF BACILLUS SUBTILIS 168
No full textAmong Gram-positive bacteria, strain 168 of Bacillus subtilis constitutes a tool of
choice for basic genetic and molecular studies. We madethe observation that this
strain exhibits an extracellular lipolytic activity. We have undertaken to characterize
the corresponding gene(s).
Shotgun cloning of Bacillus subtilis 168 DNAin E. coli yielded two types of lipasepositive
clones designated lipA and lipB. However, the lipB enzyme wasrather an
esterase, on the basis of the preferential cleavage of esters of short chain fatty acids and
of the absence of fluorescent reaction on triolein/rhodamin G medium. By multiple
Tn5 transposon inactivations, gene lipA was estimated to be about 700 basepairs long.
Both genes were inactivated in B. subtilis by reciprocal recombination with the
homologous gene disrupted in vitro by a DNA segmentcontaining an antibiotic
resistance (lipA::Km; lipB::Cm). The resulting strain expressed very little - if any -
residual extracellular lipase-esterase activity. Mapping experiments indicated that lipA
is a new locus at about 22° whereaslipB, at about 306° could correspondto an esterase
gene (estB) previously described.
Sequencing of genelipA is in progress. Besides, we are seeking conditions to
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optimize the yield of extracellular lipase (culture conditions, genetic background,
transcription signals) as a prerequisite for its large scale purification
ASSESSMENT OF CATALYST-MODIFIED CONDUCTING POLYMERS FOR THE DEVELOPMENT OF AMPEROMETRIC DEHYDROGENASE ELECTRODES
No full textRedoxreactions at mediator-modified electrode surfaces have to be optimized for the development of
amperometric enzyme electrodes. This is of special importance for dehydrogenaseelectrodes, where
the electrochemical reoxidation of the reduced cofactor NADH via a covalently bound mediatorhas to
occur avoiding intermediate radicals. Recently, we have described the synthesis and the electrocatalytic
properties of copolymers of pyrrole and 1-(1-pyrrolo)-2,4,5-trichloro benzoquinonefor the mediated
oxidation of NADH (1). In this case, the electrocatalytic activity of the catalyst-modified electrode surface,
its long-term stability, the porosity and morphology of the conducting polymerfilm and the adhesion
on the electrode surface have to be evaluated and enhanced. Until now, the electrocatalytic activity
of modified electrode surfaces has been investigated by meansofcyclic voltammetry or constantpotential
chronoamperometry. However, the enzyme, necessary coenzymes, and/or mediators have to
be present dissolved in the electrolyte, and hence sucha solution could only be used once. Especially
with very expensive enzymes orthose which aredifficult to isolate, this does not seem to be a possible
way for the optimization of electrode characteristics. Additionally, the simple physical immobilization of
dissolved enzymesin front of the electrode surface by meansof a dialysis membrane doesnotlead to
a reproducible amount andactivity of the enzymewithin the electrode chamber. Onthe other hand, the
application of enzyme reactors with enzymescovalently bound on controlled porousglass is well
knownin flow-injection systems. These enzyme-modified glass can be obtainedeasily, the activity of
the thus immobilized enzymesis in generalstabilized, and the enzyme activity/mg glass can be
determined by conventional Spectrophotometric activity assays.
In this communication, the application of dehydrogenases covalently bound to controlled-porous glass
and physically retained by meansof a dialysis membranein front of a chloranil-modified conducting
polymerelectrodeis investigated. By this method, a reproducible amountof enzymecan be kept near
the catalytically active electrode, and thus amperometric dehydrogenaseelectrodes can be compared
with respect to the electrocatalytic properties of the redox polymerfilm
A COULOMETRIC SENSOR - ACTUATOR-SYSTEM FOR ENZYMATIC MEASUREMENTS WITH LONG TERM STABILITY
No full textRapid and precise working microcoulometric flow
titrations are suitable to follow enzyme catalyzed
reactions producing ammonia. Ammonia was separated from
the effluent of enzyme microreactors or directly from
the enzyme immobilisate by an almost quantitatively
working gas dialysis step. Highly precise and accurate
determinations of urea, glutamine, asparagine and
ammonia were implemented.
A compact and fully automated microcoulometric sensor -
actuator system for fast and long term stable enzymatic
determinations was developed. The new measuring device
allows nearly absolute determinations. Therefore the
frequency of recalibration can be decreased
considerably
Development and Applications of a Four-Channel Enzyme Thermistor System for Bioprocess Control
No full textA large number of papers on biosensors have been
published in the last few years. However, few of these sophisticated
analysis systems have been used to monitor real bioprocesses. In this
paper, a newly developed four-channel enzyme thermistor system and
its application for biotechnological process monitoring is presented
and discussed. Different sugars were detected simultaneously and online
during the cultivation of Spodoptera frugiperda and Bacillus
licheniformis in technical media. Immobilized enzymes and entrapped
microorganisms were used as biological compoundin this biosensor.
In addition, enantioselective analysis was performed by two enzyme
reactions. For example, the detection of D,L-racemates of aminoacid
esters was presented in an aqueous system. Futhermore the possibility
of using this detection system in organic solvents was shown
DESIGN, FABRICATION AND MEASUREMENTOFA FET TRANSDUCER FOR CHEMICAL SENSORS
No full textA FET Transducer for chemical sensors with the focus on experimental work was designed and
fabricated using a standard NMOS, aluminum gate fabrication process. The time consuming
packaging procedure is avoided by using a teflon sample holder, which allows to contact the
device via needle probes. First measurements were made to proof the suitability of the designed
transistors for further research work