Covalent coupling method for lipase immobilization on controlled pore silica in the presence of nonenzymatic proteins

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica previously activated with glutaraldehyde in the presence of nonenzymatic proteins. This strategy is suggested to protect the enzyme from aggregation effects or denaturation that occurs as a result of the presence of silane precursors used in the formation of the silica matrix. The immobilization yield was evaluated as a function of the lipase loading and the additive type (albumin and lecithin) using statistical concepts. In agreement with the mathematical model, the maximum coupling yield (32.2%) can be achieved working at high lipase loading (450 units x g(-1) support) using albumin as an additive. In these conditions, the resulting immobilized lipase exhibits high hydrolytic (153.2 U x mg(-1)) and esterification (337.6 mmol x g(-1) x min) activities. The enhanced activity of the final lipase derivative is the sum of the benefits of the immobilization (that prevents enzyme aggregation) and the lipase coating by additives that increases the accessibility of active sites to the substrate.     

Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures]

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.     

A preliminary study of a biosensor based on flow injection of the recognition element

A biosensor based on flow injection of the recognition element has been developed. As a model a pH-transducer was used, and urease was chosen as the recognition element. The pH-transducer was immersed in an internal flow-through chamber which was in contact with the sample solution via a semi-permeable membrane. The recognition element, urease, was injected into the buffer solution passing through the biosensor. The enzyme catalysed the hydrolysis of urea and the concomitant increase in pH was recorded. The biosensor response time was about three minutes at a constant flow rate of 0·05 ml/min. The linear range of the calibration curve of the biosensor was 0–5 mM. The observed detection limit was approximately 0.1 mM. The sample throughput was 6–12 per hour. The pH-response of the biosensor, for a sample solution containing urea (3·26 mM), showed a reproducibility (r.s.d) of 28% (n = 5) and a repeatability (r.s.d.) of 8% (n = 5). Operation at elevated temperatures (up to 50°C) was demonstrated. The presence of glucose (28 mM), acetone (6·7 mM), citric acid (0·2 mM) or sodium acetate (0·6 mM) in the sample solution did not interfere with the sensor response. A lowering of the biosensor response which was observed in the presence of copper ions (due to urease inhibition) could be completely eliminated by adding EDTA to the urease solution. Thus, this work demonstrates a new type of biosensors, based on SIRE-technology (Sensors with Injectable Recognition Elements), which show high accuracy and stability, quick response and high sample throughput. These features suggest the suitability of the system for automation. Such sensors should readily be combined with other enzymes or enzyme systems. The enzyme (urease) cost per analysis (injection) for the biosensor was estimated to be approximately US$0·02. This could be substantially reduced by further optimisation and miniaturisation.     

Use of a biosensor to determine the binding kinetics of five lectins for Galactosyl-N-acetylgalactosamine

The dietary lectins, edible mushroom (ABL) and Jacalin (JAC) inhibit the proliferation of colonic cancer cells, whereas Amaranth (ACL) and peanut (PNA) stimulate their proliferation. All these lectins share as their preferred ligand the Thomsen-Friedenreich (TF) antigen galactosyl 1,3 N-Acetylgalactosamine (Gal1,3GalNAc), but differ in their finer specificities for modifications of this determinant and in their specificities for cancerous epithelia. We have investigated, using a resonant mirror biosensor, the kinetics of binding of these lectins, and Maclura pomifera lectin (MPL), which is similar to JAC, to two different Gal-GalNac bearing glycoproteins, antarctic fish antifreeze glycoprotein (AFG) and asialofetuin. JAC had the highest affinity for AFG [K _d 0.027 M] due to a fast association rate constant [k _ass 610,000 (Ms)^–1]. The other lectins had considerably lower affinities, with K _d ranging from 0.16 M (ABL) to 5.7 M (PNA), largely due to slower k _ass [ABL 74,000 (Ms)^–1 to PNA 2700 (Ms)^–1]. Similarly, JAC had a much higher affinity for asialofetuin [K _d 0.083 M] than the other lectins [K _d 1.0 M–4.5 M]. Affinities were also calculated from the extent of binding at equlibrium and were generally similar to those calculated from the kinetic parameters indicating the true nature of these values.     

Development of a conductimetric biosensor using immobilised Rhodococcus ruber whole cells for the detection and quantification of acrylonitrile

A conductimetric biosensor for the detection of acrylonitrile in solution was designed and characterised using whole cells of Rhodococcus ruber NCIMB 40757, which were immobilised into a disc of dimethyl silicone sponge (ImmobaSil). The biosensor described was capable of the detection and quantification of acrylonitrile in aqueous solution, having a linear response to concentrations between 2 and 50 mM (106-2650 ppm) acrylonitrile. The biosensor has been shown to be reproducible with respect to the data obtained over a number of days, and retains stability for a minimum period of at least 5 days before recalibration of the biosensor is required.     

A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors

An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass (CPG) beads. Soluble receptor--3H-steroid complex (cytosol or nuclear extract) is adsorbed quantitatively within the crevasses of porous glass beads. Excess labeled steroid as well as most non-specifically bound steroid is easily washed away, leaving the hormone-receptor complex retained by the beads. Bound 3H-steroid is eluted with ethanol and measured for radioactivity. This procedure which is simple, rapid, and highly reproducible is carried out using frozen samples (stable for many months) containing as few as 1 X 10(7) cells. A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration, while the CPG and whole cell assays provide equivalent values for total receptor content.     

Enzymatic synthesis of tricaprylin in a solvent-free system: lipase regiospecificity as controlled by glycerol adsorption on silica gel

Enzymatic synthesis of tricaprylin from caprylic acid (octanoic acid) and glycerol was carried out under solvent-free conditions. Kinetics of mono-, di- and tricaprylin synthesis and caprylic acid esterification in standard conditions (free glycerol) were slightly different from those obtained in the case of silica gel adsorbed glycerol. The critical role of the silica gel which behaves as a "reservoir" is proposed and it was shown that the use of silica gel facilitates reactions and improves the conversion yields at high glycerol concentrations.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

Water‐compatible silica sol–gel molecularly imprinted polymer as a potential delivery system for the controlled release of salicylic acid

Molecularly imprinted polymers (MIPs) for salicylic acid were synthesized and evaluated in aqueous environments in the aim to apply them as drug delivery carriers. One organic MIP and one inorganic MIP based on the sol–gel process were synthesized. The organic MIP was prepared by radical polymerization using the stoichiometric functional monomer, 1‐(4‐vinylphenyl)‐3‐(3,5‐bis(trifluoromethyl)phenyl)urea, which can establish strong electrostatic interactions with the –COOH of salicylic acid. The sol–gel MIP was prepared with 3‐(aminopropyl)triethoxysilane and trimethoxyphenylsilane, as functional monomers and tetraethyl orthosilicate as the crosslinker. While the organic MIPs bound the target specifically in acetonitrile, they exhibited lower binding in the presence of water, although the imprinting factor increased under these conditions, due to reduced non‐specific binding. The sol–gel MIP has a high specificity and capacity for the drug in ethanol, a solvent compatible with drug formulation and biomedical applications. In vitro release profiles of the polymers in water were evaluated, and the results were modelled by Fick's law of diffusion and the power law. Analysis shows that the release mechanism was predominantly diffusion‐controlled. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 microM.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Applying heat and mass balance theory to determine the flow rate for the measurement of benthic material flux in a flow-through system

Movement of water overlying the sediments has not been taken into consideration in most of the experiments conducted to estimate the dissolved material flux from the sediment. Even in recent experiments that incorporated the stirring motion, interpretation of the data is difficult, because the mixing rate used may be different from actual mixing rate in the field. We propose a method to estimate the in situ mixing rate that should be used to set the flow rate in a flow-through core incubation system. The flow rate is calculated from the vertical mixing rate of the water that is deduced from the heat diffusivity. Release rates of NO₃+NO₂–N obtained from our flow-through incubation system were higher by 1–3 orders of magnitude than those from the conventional diffusion calculation method that estimates the flux from the gradient of nutrient concentration across the sediment-water interface. Increase in NO₃+NO₂–N flux is considered to be due to intensification of the nitrification process as a result of an increase in dissolved oxygen (DO) supply with the motion of water. DO supply is also considered to be an important factor controlling macrofaunal abundance and consequently their excretory contributions to the fluxes of dissolved organic nitrogen as well as NH₄–N. From this point of view, we strongly recommend the application of heat and mass balance theory to estimate nitrogen flux using a flow-through experimental system.     

Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.     

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.     

Comparative study of hydrogel-immobilized l-glutamate oxidases for a novel thick-film biosensor and its application in food samples

Novel, thick-film biosensors have been developed for the determination of l-glutamate in foodstuffs. The sensors were prepared by immobilization of l-glutamate oxidase by using polycarbamylsulfonate-hydrogel on a thick-film sensor. l-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to l-glutamate at different conditions and for their specificity, inhibition, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples and show good correlation with colorimetric method.