Liquid photo-polymerized compositions as an immobilized matrix of biosensors

Series of liquid photopolimerized compositions based on oligourethanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemethacrylate (OCM-2), butilmethacrylate, methacrylic that acid, monomethacrylic ether of ethylene glycol and vinylpirrolidone (VP) were tested. It was shown the optimal variant of enzyme sensor development was a composition containing VP (a basic hydrophylic matrix), OCM-2 (crosslinked components) and OUM-2000T (crosslinked and increasing adsorption of polymer component). The blend contains 3% of enzyme. The obtained biosensors as based on immobilized beta-glucose oxidase and ureases have the following charachteristics: the linear response in the range of the concentration 0.1-10 mM, 0.05-20 mM, angle of slope of curve 30 mV/pC, 38 mV/pC, and response time 10-15, 5-10 mines, respectively. The maximal response of urease sensor was in the diapazon of pH 6.0-6.5. The increase of NaCl concentration in the solution to 300 mM caused reduction of sensor response. Under this concentration the was latter equal to half of initial response. Further increase of NaCl concentration (to 500 mM) doesn't lead to further response reduction. K(m) was calculated and it was shown, that amount of immobilized urease and beta-glucose oxidase in photopolymer material was equal 0.85 and 3.1 mM respectively.     

Photocurrent genration by thylakoid membranes immobilized in an albumin-glutaraldehyde cross-linked matrix

Summary Thylakoid membranes immobilized in an albumin-glutaraldehyde cross-linked matrix were used for photocurrent generation by a photoelectrochemical cell in potentiostatic mode. This type of preparation was quite suited for such application because it retains a substantial volume of electrolyte within the porous network formed. This property allowed for introducing electron transfer inhibitors and artificial electron acceptors and further it permitted proper migration of electroactive species from the thylakoid membranes to the working electrode as required for efficient photocurrent generation.     

Removal of heavy metals by an Aspergillus terreus strain immobilized in a polyurethane matrix

AIMS: The aim was to investigate the biosorption of chromium, nickel and iron from metallurgical effluents, produced by a steel foundry, using a strain of Aspergillus terreus immobilized in polyurethane foam. METHODS AND RESULTS: A. terreus UFMG-F01 was immobilized in polyurethane foam and subjected to biosorption tests with metallurgical effluents. Maximal metal uptake values of 164.5 mg g(-1) iron, 96.5 mg g(-1) chromium and 19.6 mg g(-1) nickel were attained in a culture medium containing 100% of effluent stream supplemented with 1% of glucose, after 6 d of incubation. CONCLUSIONS: Microbial populations in metal-polluted environments include fungi that have adapted to otherwise toxic concentrations of heavy metals and have become metal resistant. In this work, a strain of A. terreus was successfully used as a metal biosorbent for the treatment of metallurgical effluents. SIGNIFICANCE AND IMPACT OF THE STUDY: A. terreus UFMG-F01 was shown to have good biosorption properties with respect to heavy metals. The low cost and simplicity of this technique make its use ideal for the treatment of effluents from steel foundries.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

Thymidine phosphorylase activity of anucleate minicells of Escherichia coli immobilized in an agarose gel matrix

Escherichia coli whole cells, and minicells were immobilized on columns in an agarose gel matrix and evaluated for their thymidine phosphorylase (TPP) activity. The greatest specific activity of TPP was found with the induced minicells (2.2 moles thymine/min/mg protein) at a column retention time of 11 min.     

Double-stranded DNA liquid-crystalline dispersions as biosensing units

Three different approaches to constructing biosensing units based on double-stranded (ds) DNA molecules, capable of detecting various biologically active compounds, are considered. The first approach is based on the abnormal optical activity of the liquid-crystalline dispersion formed from ds DNA molecules, modified by relevant physical factors or treated with biologically active compounds. The second one is based on the abnormal optical activity of the liquid-crystalline dispersions formed first from the ds DNA and then treated with coloured biologically active compounds. The third one is based on the abnormal optical activity, specific to particles of the liquid-crystalline dispersions, where the neighbouring DNA molecules are crosslinked by artificial polymeric bridges. These approaches permit the detection of biologically relevant compounds of various origins.     

An immobilized Thiobacillus thioparus biosensing system for monitoring sulfide hydrogen; optimized parameters in a bioreactor

A microbial biosensing system for detection of hydrogen sulfide has been developed by using immobilized Thiobacillus thioparus TK-m in poly vinyl alcohol matrix, together with a dissolved oxygen sensor. Parameters of immobilization (poly vinyl alcohol concentration and amount of wet cell) were optimized by using statistical software. The obtained values for concentration of poly vinyl alcohol and wet cell weight were 11.3% (w/v) and 45 mg, respectively, where the response time of biosensor was 80 s. Calibration of oxygen concentration based on hydrogen sulfide concentration was investigated between 1 mg/L and 20 mg/L. The effect of pH and temperature were investigated in specific range of experimental conditions as well. Some parameters including operational stability and detection limit were studied in detail for characterization of biosensing system. In order to determine the operational stability, bio-sensing system at optimized working conditions was used to distinguish viability of microorganisms in polymer beads in period of time.     

Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

Properties of thermolysin immobilized in polymer matrix by radiation polymerization

Thermolysin (Bacillus thermoproteolyticus neutral proteinase, EC 3.4.24.4) has been immobilized by radiation polymerization of hydrophilic and hydrophobic monomers, and its properties, such as enzyme activity, thermal stability and durability, have been studied. The activity of the immobilized enzymes increased with an increase in the hydrophilicity of the polymer matrix and with a decrease in monomer concentration. Immobilization with hydrophilic monomers increased the thermal stability of the enzymes, but the thermal stability of the enzymes immobilized with hydrophobic monomers was comparable with that of native enzymes. The durability of the immobilized enzymes was examined by continuous hydrolysis of casein; enzymes immobilized with a high concentration (90%) of hydrophilic monomers appeared to be stabilized and could be used for long times.     

Application of microbial cells as multistep catalysts in potentiometric biosensing electrodes

The advantages of using intact microbial cells to mediate complex reaction sequences in the construction of biosensing electrodes are domonstrated. A potentiometric sensor was developed for nitrilotriacetic acid (NTA) in which a strain of Pseudomonas sp. was coupled to an ammonia gas-sensing electrode. the bacterial cells carry out a four-step reaction sequence to produce the measured species, ammonia, from NTA. The resulting microbial electrode responded to NTA in the concentration range of 1 × 10^?4 M to 7 × 10^?4M, with a slope of 35-40 mV/decade. The electrode was stable for up to one month.     

Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix**

Lipases from Pseudomonas cepacia (P.c.) and Thermomyces lanuginosa (T.l.) were immobilized in a phyllosilicate sol-gel matrix and studied for their ability to catalyze the alcoholysis of fats and oils to simple alkyl esters. At 50 degrees C and 48 h reaction immobilized T.l. lipase gave higher alkyl ester yields (70 to 100%) from fats and oils regardless of chain length or degree of unsaturation of the acyl groups in the triacylglycerols than did immobilized P.c. lipase (20-90%), which preferred unsaturated oils. Both immobilized lipases catalyzed ester formation (80-90%) from greases containing a range of free fatty acids (2.6 to 36%). Molecular sieves had no effect on ester yields in the immobilized T.l. lipase-catalyzed alcoholysis of greases but did improve yields (5-10%) in the immobilized P.c. lipase-catalyzed reactions.     

Polymethylglutamate as a new matrix for covalently immobilized enzymes: Preparation and properties of urease and uricase

Polymethylglutamate (PMG), a synthetic polypeptide, was used as a new carrier to immobilize urease (EC 3.5.1.5) and uricase (EC 1.7.3.3) by the azide method. The enzymes could be immobilized onto PMG in various forms, such as film, fiber, coating on various beads, and a silicon tube. The retained activities of the immobilized enzymes were excellent (more than 95%), therefore it was possible to immobilized almost all activities of the enzymes added in the coupling mixtures. Heat stabilities of the resulting immobilized enzymes were markedly improved, while the optimal pH and K_m values remained almost unchanged. The urease immobilized on the PMG-coated glass beads packed in a column, was found to retain its activity more than 80% of the initial value, even after the occasional use for a year. In view of the improved retained activities and stabilities of the immobilized enzymes, PMG may therefore be a very versatile matrix for the immobilized enzymes.     

Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin we prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.     

Continuous ethanol production by yeast cells immobilized in open pore gelatin matrix

Summary Open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of Ca alginate from the composite matrix followed by crosslinking with glutaraldehyde. Saccharomyces uvarum cells immobilized in the porous carrier showed high ethanol productivities with a maximum value of 25 g/l.h when monitored in packed bed reactors at 35°C with continuous cane molasses feedstock containing 10% fermentable sugars.     

Screen-printed electrodes modified with glucose oxidase immobilized in hybrid organosilicon sol-gel matrix

A new polymer composition was developed based on hybrid sol-gel material for the immobilization of enzymes on the surface of screen-printed carbon electrodes modified with Prussian blue. The existing and developed methods for the glucose oxidase (GO) immobilization are compared. Highly stable bioelectrodes were shown to be obtained by enzyme immobilization on a hybrid composition consisting of solgel/poly vinyl alcohol (PVA) (up to 60 sequential chains) and agar gel (up to 45 sequential chains). The range of glucose concentrations detected during enzyme immobilization in a hybrid sol-gel/PVA composition or agar gel without dilution of a sample was 1.0–5.9 μM and 3.6–6.3 μM, respectively. An analysis of wine products was conducted. The results obtained using the proposed biosensors were shown to differ insignificantly from those obtained by high-performance liquid chromatography (the correlation coefficient was 0.9998).     

DNA immobilized onto an acyl-azide derivative of collagen membranes for use as immunoadsorbent

Single- and double-stranded DNA were immobilized on films of highly polymerized collagen by a covalent process. The coupling was efficient at an acidic pH with an optimum at pH 5, while preventing the collagen film from any damage. In addition, no leakage occurred, so it was possible to use this DNA-coated collagen film as an immunoadsorbent. Therefore the findings reported here suggest that the acyl-azide procedure is also suitable for DNA binding on a proteinic support. Promising results in specific clearance of DNA antibodies were obtained in vitro.     

Sedation of an original data matrix as applied to paleoecology

Seriation of an original data matrix provides the paleoecologist with a simple and powerful tool for data manipulation. The data matrix lists the presences and absences of taxa in samples. The strategy is to generate a unidimensional sequence by rearranging the taxa and samples so that the presences are blocked along the matrix diagonal. The method is illustrated with two case studies from the Middle Devonian Hamilton Group of New York. The first represents a regressive interval and the seriation sequence summarizes the changes in fauna and samples which can be explained by falling sea level. The second example embraces a much wider range of variation. Seriation mainly recovers a first-order pattern related to depth and correlated variables. This seriation does not reveal parameters such as the nature of the substrate, turbidity, habitat stability and distance offshore. These data cannot be reduced to one dimension. In all cases, seriation yields results similar to those of cluster analysis and the first axis of multivariate ordinations frequently used by ecologists and paleoecologists. Seriation, paleoecology, Devonian, Hamilton Group, multivariate analysis .