Cross-linked enzyme aggregates Dotaz Zobrazit nápovědu
An amperometric biosensor compatible with a flow injection analysis (FIA) for highly selective determination of acetaminophen (APAP) in a sample of human urine was developed. This biosensor is also suitable for use in the routine pharmaceutical practice. To prove this statement, two different commercially available pharmaceutical formulations were analyzed. This nano-(bio)electroanalytical device was made from a commercially available screen-printed carbon electrode covered by a thin layer of non-functionalized graphene (NFG) as amperometric transducer. A biorecognition layer was prepared from mushroom (Agaricus bisporus) tyrosinase (EC 1.14.18.1) cross-linked using glutaraldehyde, where resulting aggregates were covered by Nafion®, a known ion exchange membrane. Owing to the use of tyrosinase and presence of NFG, the developed analytical instrument is able to measure even at potentials of 0 V. Linear ranges differ according to choice of detection potential, namely up to 130 μmol L-1 at 0 V, up to 90 μmol L-1 at -0.1 V, and up to 70 μmol L-1 at -0.15 V. The first mentioned linear range is described by the equation Ip [μA] = 0.236 - 0.1984c [μmol L-1] and correlation coefficient r = 0.9987; this equation was used to quantify the content of APAP in each sample. The limit of detection of APAP was estimated to be 1.1 μmol L-1. A recovery of 96.8% (c = 25 μmol L-1, n = 5 measurements) was calculated. The obtained results show that FIA is a very selective method for APAP determination, being comparable to the chosen reference method of reversed-phase high-performance liquid chromatography.
- MeSH
- Agaricus enzymologie MeSH
- analýza moči přístrojové vybavení metody MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- design vybavení MeSH
- lidé MeSH
- limita detekce MeSH
- neopioidní analgetika moč MeSH
- paracetamol moč MeSH
- průtoková injekční analýza přístrojové vybavení metody MeSH
- tyrosinasa chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
The enzyme immobilization is a very important process providing an attachment of the enzyme to a certain type of the carrier. This allows to maintain the enzyme activity during the storage and it also simplifies the detection procedure. When designing a new detector based on the enzymatic reaction, the proper immobilization method must be chosen depending on the advantages and disadvantages of the available methods of immobilization. Examples of immobilization method for colorimetric detection include, e.g., adsorption to the insoluble carrier, covalent bonding, attachment to ion exchangers, incorporation into gels and foams, immobilization in the form of cross-linked aggregates or nanostructures or with the utilization of antibodies.
- Klíčová slova
- kovalentní vazba,
- MeSH
- adsorpce MeSH
- cholinesterasové inhibitory MeSH
- cholinesterasy MeSH
- enzymy * MeSH
- gely analýza chemie MeSH
- imobilizace * metody přístrojové vybavení MeSH
- Publikační typ
- práce podpořená grantem MeSH
Ligninolytic enzymes from white-rot fungi are widely used in biotechnological processes. However, the application of these enzymes as free enzymes is limited due to their instability and lack of reusability. Enzyme stabilization is therefore a major challenge in biocatalytic process research, and immobilization methods are desirable. Using cross-linked enzyme aggregates (CLEAs) such as magnetic CLEAs, porous-CLEAs and combi-CLEAs is a promising technique for overcoming these issues. Cross-linking methods can stabilize and immobilize enzymes by interconnecting enzyme molecules via multiple bonds using cross-linking agents such as glutaraldehyde. The high catalyst density and microporous assembly of CLEAs guarantee high catalyst activity, which, together with their long shelf life, operational stability, and reusability, provide a cost-efficient alternative to matrix-assisted immobilization approaches. Here, we review current progress in ligninolytic enzyme immobilization and provide a comprehensive review of CLEAs. Moreover, we summarize the use of these CLEAs for biocatalysis processes, bioremediation such as dye decolourization, wastewater treatment or pharmaceutically active compound elimination.
The key to obtaining an optimum performance of an enzyme is often a question of devising a suitable enzyme and optimisation of conditions for its immobilization. In this study, laccases from the native isolates of white rot fungi Fomes fomentarius and/or Trametes versicolor, obtained from Czech forests, were used. From these, cross-linked enzyme aggregates (CLEA) were prepared and characterised when the experimental conditions were optimized. Based on the optimization steps, saturated ammonium sulphate solution (75 wt.%) was used as the precipitating agent, and different concentrations of glutaraldehyde as a cross-linking agent were investigated. CLEA aggregates formed under the optimal conditions showed higher catalytic efficiency and stabilities (thermal, pH, and storage, against denaturation) as well as high reusability compared to free laccase for both fungal strains. The best concentration of glutaraldehyde seemed to be 50 mM and higher efficiency of cross-linking was observed at a low temperature 4 °C. An insignificant increase in optimum pH for CLEA laccases with respect to free laccases for both fungi was observed. The results show that the optimum temperature for both free laccase and CLEA laccase was 35 °C for T. versicolor and 30 °C for F. fomentarius. The CLEAs retained 80% of their initial activity for Trametes and 74% for Fomes after 70 days of cultivation. Prepared cross-linked enzyme aggregates were also investigated for their decolourisation activity on malachite green, bromothymol blue, and methyl red dyes. Immobilised CLEA laccase from Trametes versicolor showed 95% decolourisation potential and CLEA from Fomes fomentarius demonstrated 90% decolourisation efficiency within 10 h for all dyes used. These results suggest that these CLEAs have promising potential in dye decolourisation.
- MeSH
- azosloučeniny chemie MeSH
- barva MeSH
- barvicí látky chemie MeSH
- bromthymolová modř chemie MeSH
- enzymy imobilizované chemie MeSH
- glutaraldehyd chemie MeSH
- katalýza MeSH
- lakasa chemie MeSH
- Polyporales enzymologie MeSH
- reagencia zkříženě vázaná chemie MeSH
- rosanilinová barviva chemie MeSH
- síran amonný chemie MeSH
- teplota MeSH
- Trametes enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Enzymes Are Powerful and Highly Specific Catalysts 140 -- Substrate Binding Is the First Step in Enzyme States 141 -- Enzymes Can Use Simultaneous Acid and Base Catalysis 144 -- Lysozyme Illustrates How an Enzyme - Peplication in the 5\'-to-3\' Direction Allows Efficient Correction -- \\jcleotide-Polymerizing Enzyme Analysis 491 -- Rapid and Cheap DNA Sequencing Has Revolutionized -- Human Genetic Studies 491 -- Linked Accept and Release Electrons Readily -- NADH Transfers Its Electrons to Oxygen Through Three Large Enzyme
Sixth edition xxxiv, 1430 stran v různém stránkování : ilustrace (převážně barevné) ; 29 cm
"As the amount of information in biology expands dramatically, it becomes increasingly important for textbooks to distill the vast amount of scientific knowledge into concise principles and enduring concepts. As with previous editions, Molecular Biology of the Cell, Sixth Edition accomplishes this goal with clear writing and beautiful illustrations. The Sixth Edition has been extensively revised and updated with the latest research in the field of cell biology, and it provides an exceptional framework for teaching and learning. The entire illustration program has been greatly enhanced. Protein structures better illustrate structure-function relationships, icons are simpler and more consistent within and between chapters, and micrographs have been refreshed and updated with newer, clearer, or better images. As a new feature, each chapter now contains intriguing open-ended questions highlighting "What We Don't Know," introducing students to challenging areas of future research. Updated end-of-chapter problems reflect new research discussed in the text. Thought-provoking end-of-chapter questions have been expanded to all chapters, including questions on developmental biology, tissues and stem cells, the immune system, and pathogens"--Provided by publisher.
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- molekulární biologie, molekulární medicína
- NLK Publikační typ
- učebnice vysokých škol
Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques - bright-field, confocal and electron microscopy - were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non-treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions.
- MeSH
- biokatalýza MeSH
- dioxany farmakologie MeSH
- furany farmakologie MeSH
- hydrolasy chemie metabolismus MeSH
- mikroskopie metody MeSH
- proteiny červů chemie metabolismus MeSH
- Schistosoma japonicum enzymologie MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glycation is a process closely related to the aging and pathogenesis of diabetic complications. In this process, reactive α-dicarbonyl compounds (e.g., methylglyoxal) cause protein modification accompanied with potential loss of their biological activity and persistence of damaged molecules in tissues. We suppose that glutathione S-transferases (GSTs), a group of cytosolic biotransformation enzymes, may be modified by glycation in vivo, which would provide a rationale of its use as a model protein for studying glycation reactions. Glycation of GST by methylglyoxal, fructose, and glucose in vitro was studied. The course of protein glycation was evaluated using the following criteria: enzyme activity, formation of advanced glycation end-products using fluorescence and western blotting, amine content, protein conformation, cross linking and aggregation, and changes in molecular charge of GST. The ongoing glycation by methylglyoxal 2 mM resulted in pronounced decrease in the GST activity. It also led to the loss of 14 primary amino groups, which was accompanied by changes in protein mobility during native polyacrylamide gel electrophoresis. Formation of cross links with molecular weight of 75 kDa was observed. Obtained results can contribute to understanding of changes, which proceed in metabolism of xenobiotics during diabetes mellitus and ageing.
- MeSH
- diabetes mellitus enzymologie MeSH
- fruktosa chemie metabolismus MeSH
- glukosa chemie metabolismus MeSH
- glutathiontransferasa chemie metabolismus MeSH
- katalýza MeSH
- lidé MeSH
- produkty pokročilé glykace chemie metabolismus MeSH
- pyruvaldehyd chemie metabolismus MeSH
- stárnutí metabolismus MeSH
- xenobiotika chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
activating thrombin activatable fibrinolysis in- -- hibitor (TAFI) and factor XIII (FXIII), the terminal enzyme are activated by serine -- proteases of the blood coagulation cascade, notably thrombin, and are -- linked Discussion is restricted to the main im- -- aging modalities used in these tumors: cross-sectional imaging Additional platelets form aggregates on those platelets that have -- adhered to the vessel wall.
svazky
- MeSH
- dítě MeSH
- krevní nemoci MeSH
- novorozenec MeSH
- Check Tag
- dítě MeSH
- novorozenec MeSH
- Publikační typ
- sborníky MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- onkologie
- hematologie a transfuzní lékařství
Self-assembling Aggregates Can Include Different 87 Protein Subunits and Nucleic Acids -- 91 -- 92 - Their Plasma Membrane Proteins Rapidly Mix -- Membrane Proteins Cluster into Patches When They Are Cross-linked by Antibodies -- Cross-linked Membrane Proteins Are Actively Swept to One Pole of the Cell in the Process Proteins in Nonmuscle Cells 582 -- Microvilli Contain Bundles of Actin Filaments 582 -- Rigid Arrays of Cross-linked 699 -- Elastin Is a Cross-linked, Random-Coil Protein That -- Gives Tissues Their Elasticity 701 --
xxxix, 1146 s. : il., tab. ; 28 cm
- MeSH
- biologie buňky MeSH
- molekulární biologie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- cytologie, klinická cytologie