Determination of concentration and activity of immobilized enzymes

Singh, Priyanka; Morris, Holly S.; Tivanski, Alexei V.; Kohen, Amnon

doi:10.1016/j.ab.2015.02.014

Cited by 8 publications

(16 citation statements)

References 24 publications

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“…Despite the benefits of immobilization from the processing viewpoint, a major concern is to assure that, upon immobilization, no detrimental changes are induced in the secondary and tertiary structure of the enzyme molecules [ 27 , 28 ]. These detrimental changes are responsible for altering the active sites and consequently, for a significant reduction in the catalytic competency of the prepared immobilizates [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

Congo Red Decolorization Using Textile Filters and Laccase-Based Nanocomposites in Continuous Flow Bioreactors

et al. 2020

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Removal of azo and diazo dye content from textile industry wastewaters is crucial due to their environmental impact. Here, we report on the use of the fungal laccase from Pycnoporus sanguineus CS43 immobilized on silica nanoparticles and entrapped in textile-based filters for the degradation of Congo Red. Laccase immobilization and synthesis of the nanocomposites were carried out by two different methods, one in the presence of acetone and the second using water as solvent. This led to a change in the hydrophobicity of the obtained biofilters. Successful preparation of the nanocomposites was confirmed via FTIR spectroscopy. Changes in the secondary structure of the enzyme were inspected through the second derivative of the FTIR spectra. Six different types of filter were fabricated and tested in a continuous flow bioreactor in terms of their decolorization capabilities of Congo Red. The results indicate removal efficiencies that approached 40% for enzymes immobilized on the more hydrophobic supports. Backscattered electron (BSE) images of the different filters were obtained before and after the decolorization process. Percentage of decolorization and activity loss were determined as a function of time until a plateau in decolorization activity was reached. Experimental data was used to recreate the decolorization process in COMSOL Multiphysics® (Stockholm, Sweden). These simulations were used to determine the proper combination of parameters to maximize decolorization. Our findings suggest that the treatment of textile-based filters with immobilized laccase in conjunction with hydrophobic nanocomposites provides a suitable avenue to achieve more efficient laccase dye decolorization (39%) than that obtained with similar filters treated only with free laccase (8%). Filters treated with silica-based nanocomposites and immobilized laccases showed an increase in their decolorization capability, probably due to changes in their wetting phenomena.

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Section: Introductionmentioning

confidence: 99%

Congo Red Decolorization Using Textile Filters and Laccase-Based Nanocomposites in Continuous Flow Bioreactors

et al. 2020

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“…29,30 Amine and thiol functional 123 base pair dsDNA linkers were prepared by the polymerase chain reaction (PCR) method utilizing Taq DNA polymerase and pEt22b-DHFR vector as a template. 22 The PCR was performed with 5′ thiol_TTA GCG GTA GAT CGC GTT ATC GGC ATG and 5′ amino_TTC CCA GGT ATG GCG GCC CAT AAT CAC primers (Integrated DNA Technologies, Coralville, IA) to obtain a 41 nm long dsDNA molecule with amine and thiol functional groups in complementary ends (Scheme S1 in the Supporting Information). The dsDNA thiol end is protected with S-trityl-6mercaptohexyl protecting group to prevent the disulfide bond formation between the dsDNA molecules.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Rigid Double-Stranded DNA Linkers for Single Molecule Enzyme–Drug Interaction Measurements Using Molecular Recognition Force Spectroscopy

et al. 2020

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Single-molecule studies can reveal the distribution of states and interactions between ligand–enzyme complexes not accessible for most studies that measure a large ensemble average response of many molecules. Furthermore, in some biological applications, the information regarding the outliers, not the average of measured properties, can be more important. The high spatial and force resolution provided by atomic force microscopy (AFM) under physiological conditions has been utilized in this study to quantify the force–distance relations of enzyme–drug interactions. Different immobilization techniques of the protein to a surface and the drug to AFM tip were quantitatively compared to improve the accuracy and precision of the measurement. Protein that is directly bound to the surface, forming a monolayer, was compared to enzyme molecules bound to the surface with rigid double-stranded (ds) DNA spacers. These surfaces immobilization techniques were studied with the drug bound directly to the AFM tip and drug bound via flexible poly(ethylene glycol) and rigid dsDNA linkers. The activity of the enzyme was found to be not significantly altered by immobilization methods relative to its activity in solution. The findings indicate that the approach for studying drug–enzyme interaction based on rigid dsDNA linker on the surface and either flexible or rigid linker on the tip affords straightforward, highly specific, reproducible, and accurate force measurements with a potential for single-molecule level studies. The method could facilitate in-depth examination of a broad spectrum of biological targets and potential drugs.

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“…A partial list of references includes (Arora & Brooks III, 2009; Bystroff & Kraut, 1991; Dametto et al, 2012; Fan et al, 2013; Fierke et al, 1987; Klinman & Kohen, 2013; H. Liu & Warshel, 2007b; Loveridge, Behiry, Guo, & Allemann, 2012; McElheny, Schnell, Lansing, Dyson, & Wright, 2005; Pauling, 1948; Singh, Abeysinghe, & Kohen, 2015; Singh, Francis, & Kohen, 2015; Singh, Morris, Tivanski, & Kohen, 2015a, 2015b; Z. Wang, Singh, Czekster, Kohen, & Schramm, 2014; Kim F. Wong, Watney, & Hammes-Schiffer, 2004).…”

Section: Case Study 2 – Dihydrofolate Reductase (Dhfr)mentioning

confidence: 99%

Examinations of the Chemical Step in Enzyme Catalysis

Singh

Islam

Kohen

2016

Methods in Enzymology

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Advances in computational and experimental methods in enzymology have aided comprehension of enzyme-catalyzed chemical reactions. The main difficulty in comparing computational findings to rate measurements is that the first examines a single energy barrier while the second frequently reflects a combination of many microscopic barriers. We present here intrinsic kinetic isotope effects and their temperature dependence as a useful experimental probe of a single chemical step in a complex kinetic cascade. Computational predictions are tested by this method for two model enzymes: dihydrofolate reductase (DHFR) and thymidylate synthase (TSase). The description highlights the significance of collaboration between experimentalists and theoreticians to develop a better understanding of enzyme-catalyzed chemical conversions.

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Determination of concentration and activity of immobilized enzymes

Cited by 8 publications

References 24 publications

Congo Red Decolorization Using Textile Filters and Laccase-Based Nanocomposites in Continuous Flow Bioreactors

Congo Red Decolorization Using Textile Filters and Laccase-Based Nanocomposites in Continuous Flow Bioreactors

Rigid Double-Stranded DNA Linkers for Single Molecule Enzyme–Drug Interaction Measurements Using Molecular Recognition Force Spectroscopy

Examinations of the Chemical Step in Enzyme Catalysis

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