67 research outputs found
Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications
No full textIn this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3611
Tuning of a central controller for a sewer network using multiple simplified models
No full textWater Resource
Ultra-thin mems fabricated tynodes for electron multiplication
No full textFor decades, photomultiplier tubes (PMTs) have been the most common choice in single photon detection, covering the spectral range from deep-ultraviolet to nearinfrared. PMT is a vacuum tube with three crucial components: photocathode, chain of dynodes and anode. At the photocathode, photons are converted to electrons in a photoelectric effect, after which they are directed to the dynodes chain. The material and geometry of dynodes are chosen to efficiently amplify the charge through the secondary electron emission (in reflection mode). Finally, created avalanche of electrons is collected and measured by the anode. Timed Photon Counter (TiPC) is a novel vacuum-based photomultiplier proposed to overcome limitations of PMTs in terms of size, speed, spatial resolution and operation in the presence of magnetic field. The key novelty of TiPC is a tynode – a large-size array of ultra-thin, free-standing membranes which, in contrast to dynodes, multiply electrons in the transmission mode. Due to the short and straight crossing paths of electrons between subsequent tynodes, the time resolution of the TiPC can be in the order of 10 -12 s. The set of tynodes is placed under the photocathode, and on top of a CMOS detecting chip. With such design, TiPC represents a light, compact and ultra-fast photodetecting device with a high relevance for solid state, atomic and molecular physics experiments, medical imaging and 3D optical imaging. The focus of this thesis is microelectromechanical systems (MEMS) fabrication of the tynodes. To our knowledge, this is the first time MEMS technology is employed as a powerful tool for the production of large arrays of free-standing membranes, with thicknesses of only a few nanometers, to be used in photodetection. Detailed analysis in terms of mechanical, optical, electrical and structural properties were performed in order to discern the most suitable material for the TiPC application among the investigated candidates. The transmission SEY (TSEY) of the released tynodes is analysed with a dedicated setup, specifically developed in our group, inserted in a scanning electron microscope (SEM). Low pressure chemical vapour deposition (LPCVD) was employed as a technique to grow silicon nitride (SiN) tynodes with varied layout, elemental stoichiometry and thicknesses in the range from 25 to 40 nm. Due to its inability to produce good-quality films with thicknesses lower than 20 nm, LPCVD was replaced by atomic layer deposition (ALD). It was found that SiN performs poorly in terms of secondary electron emission (SEE), and we selected Al2O3 (alumina) as the next tynode material. The ALD of alumina is investigated in the temperature range from 300 down to 100 °C, with the goal to determine its viability in the coating of temperature-sensitive substrates such as photoresist. We demonstrated the fabrication of 5 – 25 nm-thick ALD alumina tynodes which exhibited moderately high TSEY. Apart from SiN and alumina, other materials subjected to SEE analysis in this work were: chemical vapour deposited (CVD) ultrananocrystalline diamond (UNCD), monocrystalline silicon and LPCVD silicon carbide (SiC). Applying atomic layer deposited magnesium oxide (MgO) as the tynode material resulted in a transmission secondary electron yield (TSEY) of up to 5.5, by which it proved to be the most efficient electron multiplier among materials taken into account in this work. During the fabrication of tynodes, SEE films were exposed to different MEMS processing steps, and thus inevitably undewent a surface modification which alters the SEE properties. On that account, we conducted a study on the ALD MgO films subjected to various chemical and thermal treatments and explored the methods to further enhance their SEE. For the final application in the TiPC, stacked tynodes should provide the focusing of electrons. To meet this requirement, the emission film was grown on a pre-patterned substrate, which enabled hemi-spherical shape of the released membranes. Finally, for the vertical stacking and alignment of the tynodes, steps for the formation of V-grooves were added in the standard fabrication flowchart.EKL Processin
Protein Engineering : an Option for Enzymatic Biofuel Cell Design
No full textThis review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells
A high-throughput cellulase screening system based on droplet microfluidics
No full textA new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3666
Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase
No full textSummaryGlucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme
Imobilizacija perjodatno oksidovane invertaze adsorpcijom na sepiolitu
No full textPeriodate oxidized invertase was immobilized by adsorption on sepiolite. The obtained immobilized enzyme was more resistant to washing out by concentrated salt solution. and had an eight times higher half-life at 60 degreesC than adsorbed native invertase. In packed bed reactor 50%, conversion of 500 g/dm(3) sucrose at 40 degreesC and a flow rate of 1 bv/h was achieved. The specific productivity of the immobilized invertase was 0.187 kg/dm(3)/h.Perjodatno oksidovana invertaza je imobilizovana adsorpcijom na sepiolitu. Dobijeni imobilizovani enzim je bio rezistentniji na ispiranje koncentrovanim rastvorima soli i imao je osam puta veći poluživot na 60 ºC od adsorbovane nativne invertaze. U protočnom cevastom reaktoru dobili smo 50 % konverziju rastvora saharoze koncentracije 500 g/dm3 na 40 ºC i pri protoku od 1 bv/h. Specifična produktivnost imobilizovane invertaze je bila 0,187 kg/dm3/h
Effect of cadmium stress on antioxidative enzymes during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ]
No full textWhen considering the effect of heavy metals on trees generally and on forest ecosystems especially, importance is given to their influence on seed germination in metal polluted soil. There is insufficient data on this subject, especially when conifers are concerned. In this work, the influence of high cadmium concentrations on percentage germination, specific activities and isoenzyme patterns of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ] was studied. Cadmium chloride concentrations of up to 0.1 mM did not cause an inhibition of germination, while 1 mM concentration inhibited germination and the activities of catalase, superoxide dismutase and peroxidase. The isoenzyme profile of catalase and superoxide dismutase did not change at high cadmium concentrations, while peroxidase expression of basic peroxidase (B5) with pI value of 9.1 increased. This isoform of POD can play an important role in the early development of Serbian spruce and its defense mechanism against heavy metals.Key words: Seed germination, catalase, peroxidase, superoxide dismutase, heavy metals
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