LIPASES : BIOTRANSFORMATIONS, ACTIVE SITE MODELS AND KINETICS

Lipases can be used to obtain various (chiral) intermediates. To select a suitable hydrolytic enzyme from the increasing number of commercially available lipases application of active-site models may be very useful. Since the hydrolysis takes place at the interface, the kinetics of lipase catalyzed reactions are strongly dependend upon the quantity and quality of the interface. A newly developed dynamic method, based on measuring the droplet-size distribution by light scattering (Fraunhofer diffraction), has proven to be very useful to measure the total interfacial area of a non-stabilized emulsion. In an alternative approach lipase kinetics could be determined by using a hollow fiber membrane reactor. Both approaches indicate that there is a linear relationship between the rate of lipolysis and the interfacial area. The effect of the quality of the interface on the enzymic hydrolysis reaction is currently being studied to optimize both the rate as well as the (stereo)selectivity of the hydrolysis

ELIMINATION OF INTERFERENCES IN GLUCOSE DETERMINATION IN BLOOD SERUM USING FLOW-INJECTION AMPEROMETRY

A flow-injection system with anodic amperometric detection for glucose determination in human blood serum samples was optimized by a comparison of various arrangements of enzyme immobilization and elimination of interferences such as ascorbic acid, urea and uric acid. The best results were obtained using a compact membrane biosensor in wall-jet flow-through detector, where Pt disc electrode was covered with evaporated Nafion layer, then a polyester membrane with immobilized glucose oxidase and another protective polyester membrane. In the optimized conditions a linear response up to 25mM glucose was observed with detection limit 200uM glucose and sampling rate 120 hr-1 for 20p1 sample volume. Results of flow injection glucose determination in human serum samples were compared with Beckman Glucose Analyser 2 and Kone Dynamic analyser

The Potential of Surface Acoustic Wave Devices for the Selective Detection of Trace Amounts of Molecules

Chemical Sensors and Biosensors try to mimic the capability of living systems to detect very small amounts of analyte molecules using specific host-guest type of reactions. Recent advances in microelectronic technology have led to a new generation of sensor devices, the piezoelectric microbalances, based on planar microfabrication techniques. They show a very high sensitivity ( up to femtograms ) for detecting molecules which adsorb to the surface of the device and change its resonance frequency with mass loading. All chemical or biological microsensor devices require a surface coating that will interact with the specific chemical or class of chemicals or biomolecule to be detected. The majority of applications have made use of bulk acoustic wave (BAW) devices, usually with an adsorptive coating to provide some degree of selectivity towards the analyte of interest [Glassford]. The oscillation frequency of a BAW device immersed in solution changes with the temperature of the solution, with the specific gravity and conductivity as well as the microviscosity at the interfacial layer, which can be changed by analyte binding

Simulations of the Potential Generation of an Enzyme-pH Fieldeffecttransistor (ENFET)

In order to verify the chances of the enzyme-pH fieldeffecttransistor (ENFET) for the detection of urea in practical applications, in our group computer simulations have been carried out that scrutinized the influence of various parameters on the measurement signal. The described sensor uses a glutardialdehyde membrane containing the enzyme urease and an ISFET transducer, and is immersed in a buffer solution. If urea is added to the solution, it is metabolized to carbon dioxide and ammonia. The resulting pH-increase in the The program calculates a local distribution of substrate concentration and pH-value in the membrane. The influence of the above mentioned concentrations on the pH-value (which can be regarded as the measurement signal) is discussed. Above all these simulations show that for a working enzyme-pH sensor a well defined composition of the sample solution is essential. Therefore the ENFET works best in a flow-through-system, where all parameters can be kept constant, or together with a microtitration unit. membraneis measured by the ISFET. The simulation program is based on Michaelis-Mentenrelations and on adequatediffusion-reaction-equations for the concentrations of the enzyme, the substrate, and the reaction products, as well as the buffer capacity. Also the thickness of the membrane is taken into account

APPLICATIONS AND KINETICS OF IMMOBILIZED ENZYMES AND COUPLED ENZYME REACTIONS

Immobilization of enzymesresults in more adequate reagentsto be used analytically. After immobilization, the kinetic parameters of the enzymes are modified and nothing can be predicted abouttheactivity of the heterogeneous system. In coupled enzymereactions with co-immobilized enzymes, this is even more important owingto the kinetic dependence on each consecutive reaction. The determination of ethanol and acetaldehyde is considered in this paper, using two different coupled enzyme systems. Some important parameters, as the enzyme charged,the ratio of each enzymein the sequence and the immobilization yield, are considered in terms of conversion efficiency finally determiningthesensitivity of the analysis

SIMULTANEOUS DETERMINATION OF MULTICOMPONENT IN FOOD BY AMPEROMETRIC FIA WITH IMMOBILIZED ENZYME REACTORS IN A PARALLEL CONFIGURATION

A simultaneous determination system for food components with FIAenzyme reactor methodology has been developed. The main flow lines of the system are composed of plural independent lines and plural immobilized enzyme reactors set in a parallel configuration. Enzymes used were glucose oxidase for glucose, lactate oxidase for lactate, alcohol oxidase for ethanol, g-fructosidase, mutarotase and glucose oxidase for sucrose, fructose-5-dehydrogenase for fructose, sulfite oxidase for sulfite, and glycerol dehydrogenase for glycerol. First, simultaneous determination of lactate, glucose and ethanol in alcoholic beverages and serum was performed by monitoring hydrogen peroxide. Each corresponding oxidase of the substrate was immobilized on an Amino-Cellulofine support. Interferences of ascorbate and/or urate in a sample were completely eliminated by using ascorbate- and urate-eliminating reactors. Next, glucose, fructose and sucrose were determined simultaneously. Hydrogen peroxide (for glucose and sucrose) and hexacyanoferrate (II) (for fructose) were monitored. Sucrose determination was performed with a glucose-eliminating reactor which was set just before the sucrose reactor. Interference of ascorbate was also eliminated by an ascorbate-eliminating reactor. Finally, sulfite, glucose, glycerol and ethanol in white wine were determined simultaneously. For the glycerol determination, the NADH produced was monitored at +0.75 V vs. Ag/AgCl. For sulfite determination, a platinum electrode covered with dialysis membrane was used in order to diminish the interference of polyphenol compounds

SIMULTANEOUS FLOW INJECTION ANALYSIS OF L-LACTATE AND L-MALATE IN WINE BASED ON THE USE OF ENZYMES

The 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

DIRECT OBSERVATION OF ANTI-ATRAZINE ANTIBODY BINDING USING GRATING COUPLERS

Planar 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

PROTEIN O-GLYCOSYLATION AND SEXUAL AGGLUTININS IN THE YEAST S.CEREVISIAE

The features of protein O-glycosylation in yeast are summarized. - The a and a-agglutinin of haploid S.cerevisiae cells have been purified and their one to one interaction has been studied in vitro; their carbohydrate moieties do not seem to be essential. Both the corresponding genes have been cloned and sequenced

NOVEL COVALENT AVIDIN IMMOBILIZATION ON GLASSY CARBON ELECTRODES USING A BIFUNCTIONAL REAGENT

It is generally accepted that the stability and performance of a biosensor is determined by the way biocomponents are immobilized on the inorganic support. We present a covalent coupling method of the anchor protein avidin to a glassy carbon electrode surface using the heterobifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Thus, a nearly complete, biologically active monolayer of avidin was obtained which is suitable for the immobilization of any biotinylated compound. These modified electrodes are potentially useful as amperometric biosensors