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MICROORGANISMS AND ENZYMESFOR THE APPLICATION IN BIOSENSORS
No full textCarbon monoxide, nitriles, amides, vinyl acetate, aldehydes and complex metalcyanides are examples
of toxic compounds which we are planning to detect by means of simple types of enzyme-based
biosensors, e. g. tubes, buttons, dosimeters or electrodes. Among the requirements appropriate
enzymes should meet, are the ability of being coupled to a color reaction and sensitivity in the ppm
concentration range. As appropriate enzymes were not available at the beginning of our work, we
searched for microorganisms and are currently isolating and characterizing the enzymesofinterest
from them. Apart from using these enzymes for sensing purposes weare also interested in devices
which would allow a simultaneous removal of toxic compounds. Examples discussedin this article are
a biological system for the detoxification of acetonitrile in HPLC waste and the use of enzymatically
active paint for the improvement ofindoorair quality
CLONING AND EXPRESSION OF PROTEINS FROM THERMUS THERMOPHILUS IN ESCHERICHIA COLI
No full textCloning and expression of genesfrom the extreme thermophilic bacterium Thermus
thermophilus in the mesophilic Escherichia coli was investigated. Weidentified the structural
genes (tuf1, tuf2) of the protein elongation factor Tu from Thermus thermophilus and
determined the nucleotide sequenceof tuf1. The expression of tuf1 occurs in E. coli from its T.
thermophilus own promoter. The organization of the T. thermophilus operon which contains
tuf1 is similar to the homologous operonin E. coli. The codon usage-of the T. thermophilus
genesis different from that in E. coli. Codons with a guanine in the third position are
preferentially usedin this thermophilic organism. The codon usage is mostly determined by the
necessity to stabilize the codon-anticodoninteraction at high temperature
ON-LINE MEASUREMENT OF INTRACELLULAR ENZYME ACTIVITY DURING FERMENTATION USING PERMEABILIZED CELLSIN A FIA SYSTEM
No full textA novel method for determination of intracellular enzyme activity in yeast cells has been deviced.
After permeabilization it is possible to determine the activity of NAD-dependent formate
dehydrogenase from Candida boidinii based on stopped flow injection techniques combined with
fluorimetric detection. Yeast cells were made permeable using the surfactant Digitonin in
combination with Triton X—100
CONSTRUCTIONOFAN FIA UNIT AND ITS USE IN THE INVESTIGATION OF IMMOBILISED LIGNIN PEROXIDASE
No full textA flow injection analyser was designed to serve asa tool for the characterisation of enzymes.
The original device was constructed from available laboratory equipment, and has
subsequently been refined to a fully automated, stand alone unit. The hard- and software
have been so conceived as to enable a wide range of applications. All system components
are controlled by a microprocessor, which also takes over data acquisition, peak integration
and evaluation, and provides a convenientinterface to the user via a key-board and display.
We have used this FIA system to investigate the enzymatic properties of lignin peroxidase.
This enzyme is believed to play a key role in the biodegradation oflignin by white-rot fungi,
and has attracted interest for use in various industrial processes. Lignin peroxidase has a
very similar mechanism of action to horse radish peroxidase, butdiffers in its ability to oxidise
organic compoundsof higher oxidation potential, and in its extreme sensitivity to inactivation
by excess H2O2. The enzyme can be successfully immobilised on a variety of carriers.
However when investigated in batch assays, the loss of activity is such that repeated
experimentation is impossible. FIA is characterised by small sample volumes, and thus low
contact times when working with an immobilised enzyme. We have exploited this to enable
investigation of an essentially unstable enzyme system
ON-LINE DETECTION OF LIPASE ACTIVITY BY FLOW INJECTION ZYMOGRAPHY
No full textThe application of a FIA-FPLC unit for the post-column on-line detection of lipase activity is
presented. As the development of techniques such as Fast Protein Liquid Chromatography
(FPLC) and High Performance Liquid Chromatography (HPLC)has greatly reduced the time
required for protein purification, fast identification of enzyme-containing fractions is desired.
Rapid detection of enzymeactivity can be achieved by coupling a flow injection analysis (FIA)
system to the liquid chromatographyunit. Usually lipase activity is detected in the presence of
emulsified substrates, whose even distribution in the FIA system causes problems. In order to
solve these problems, the solubilized substrates S,0,0’-tributyryl-1-thiogycerol (TBTG) and
1,2-O-dilauryl-rac-glycero-3-glutaric-resorufinester (BM) were applied to determinethe relationship
between lipase concentration and FIA response. Lipase and its substrate were injected
simultaneously into two carrier streams which were mixed together before passing a thermostated
reaction coil. The cleaved productof the lipase substrate was detected photometrically.
The BM-substrate turned out to be most suitable for FIA applications. The FPLC-FIA unit
using the BM-substrate has successfully been applied for post-column on-line monitoring of
lipase activity during differentlipase purification steps. FIA response showsa linearcorrelation
to lipase activity up to lipase concentrations of 140 U/ml
ASSESSMENT OF PROTEIN GLYCOSYLATION USING HIGH-pH ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED ELECTROCHEMICAL DETECTION
No full textAs more recombinant glycoproteins are being developed for therapeutic Purposes, there is
an increasing need for sensitive, simplified methods for analysis of their oligosaccharide
chains. High-pH anion-exchange liquid chromatography (HPAE) with pulsed amperometric
detection (PAD) has been shownto be useful in a numberof areas of structural elucidation
of glycoprotein glycans. Higher resolution of oligosaccharide mixtures, containing both neutral
and anionic components, has been reported using HPAE chromatography with micropellicular
resins. Sensitive composition analysis of both glycoproteins and oligosaccharides,
without derivatization steps, was accomplished using HPAE/PAD. Oligosaccharides were
purified in high yield using HPAE andin-line desalting through flat micro-membranes. These
purified oligosaccharides were found to be suitable for mass spectrometric and ‘H-NMR
analysis. Measurement of both released monosaccharides and changesinretention times of
purified oligosaccharides in exoglycosidase digests have been performedin a single step.
The developmentof additional AE/PAD-based methods should further simplify carbohydrate
analysis of glycoproteins
REMODELLING OF GLYCOPROTEINS WITH SIALIC ACID ANALOGUES
No full textTo complete their biosynthesis many soluble or membrane bound
glycoconjugates require a final sialylation step which is catalyzed by
distinct sialyltransferases differing in specificity with respect to
the glycan acceptor structure and the linkage formed. Important biological
functions of glycoconjugates depend on the degree of terminal
sialylation. Replacement of naturally occurring sialic acids by synthetic
analogues endowed with special properties can serve to influence
certain biological functions of soluble or membrane-bound sialoglycoconjugates.
In particular, those sialic acid analogues that
deserve special interest are sialidase-resistant or O-acetylesteraseresistant,
or they are radiolabeled, fluorescently labeled, or carry a
photoreactive functional group, or they may differ in charge or hydrophobicity
from the parent sialic acid. It has been shown that CMP
sialate synthase has a low substrate specificity with respect to the
C-5 and C-9 positions of sialic acid. As a consequence many CMPactivated
analogues of sialic acid can be obtained on a preparative
scale. Detailed studies have revealed that different sialyltransferases
accept a variety of CMP-activated sialic acid analogues as
donor substrates. This finding opens the way for preparation by
enzymatic synthesis on a large scale of glycoproteins differently
modified in the sialic acid moiety. Membrane-bound receptors can be
modified and the biological consequences studied. With accumulating
knowledge of the donor substrate specificity of CMP-sialic acid
synthase and sialyltransferase, analogues can be synthesized that are
tailored for special applications or for use as inhibitors of enzymes
of sialoglycoconjugate biosynthesis. Furthermore, glycoproteins can be
prepared that resist biological degradation by sialidases
LIPASES IN REVERSE MICELLES
No full textTwo independent spectroscopic methods have been developed to assay lipases
continuously with triacylglycerol substrates in a reverse micellar solution. With the two
methods, a simple and unique possibility is offered to study the kinetics and the
specificity of lipases, embedded in a system which possibly mimics the biologically
relevant environment of lipolytic enzymes.
Preliminary activity data are presented for the colipase dependent human pancreatic
lipase in reverse micelles, and we have started to investigate the conformational behavior
of this enzyme by means of circular dichroism and fluorescence spectroscopy
LIPASE AND WATER
No full textThe reaction rate of two lipase-catalyzed reactions, esterification and transesterification, were
studied in a liquid/solid two-phase system in orderto investigate the effect of water partition between
the enzyme preparation andthe liquid phase composedof only the reactants withoutthe conventional
solvents.
Lipase from Candida cylindracea was used for these studies. The enzyme wasinactive in
dehydrated systems. In the case of monoester synthesis, the reaction increased with increasing water
activity. The reaction rates of the lipase catalyzed reactions were very sensitive to the natere of the
substrates in this unusual system.
When transesterification and esterification were carried out in the same liquid phase, the
transesterification reaction rate was controlled by the water produced by the concomitantesterification
CLONING AND EXPRESSION OF INDUSTRIALLY IMPORTANT FUNGALLIPASES
No full textTriglyceride lipases from various organisms have a numberof potential industrial applications
exemplified by their use in flavour enhancement, in production of esters and specialty fats, and
in household detergents. We have cloned and expressed two fungallipases: the 1,3-positional
specific lipase from Rhizomucor miehei (1,2), commercialized as Lipozyme™, and lipase from
Humicola lanuginosa commercialized for use in household detergents (Lipolase™).
Production of these lipases and other enzymesforindustrial application has necessitated the
developmentof an efficient recombinant expression system. We have focusedour efforts on the
development of Aspergillus as a recombinant host system for this purpose. Aspergillus has the
capacity to secrete large amounts of active hydrolytic enzymes such as for example glucoamylases
and amylases. Cloning of strong promoters from the corresponding genes (3,4) and
development of a transformation- and selection system (4) has allowed expression from
heterologous cDNA genesin Aspergillus oryzae. As first attempt to obtain heterologous
expression we transferred an aspartic proteinase cDNA (5) from Rhizomucor miehei into A.
oryzae and obtained high levels of secreted, active and correctly processed enzyme (4). These
experiments have later been extended to fungal triglyceride lipases (6,7)