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      An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

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          Abstract

          The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.

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          Most cited references181

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          The fluorescent toolbox for assessing protein location and function.

          Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
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            Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.

            Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
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              A molecular ruler based on plasmon coupling of single gold and silver nanoparticles.

              Forster Resonance Energy Transfer has served as a molecular ruler that reports conformational changes and intramolecular distances of single biomolecules. However, such rulers suffer from low and fluctuating signal intensities, limited observation time due to photobleaching, and an upper distance limit of approximately 10 nm. Noble metal nanoparticles have plasmon resonances in the visible range and do not blink or bleach. They have been employed as alternative probes to overcome the limitations of organic fluorophores, and the coupling of plasmons in nearby particles has been exploited to detect particle aggregation by a distinct color change in bulk experiments. Here we demonstrate that plasmon coupling can be used to monitor distances between single pairs of gold and silver nanoparticles. We followed the directed assembly of gold and silver nanoparticle dimers in real time and studied the kinetics of single DNA hybridization events. These "plasmon rulers" allowed us to continuously monitor separations of up to 70 nm for >3,000 s.
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                Author and article information

                Journal
                Biotechnol Biotechnol Equip
                Biotechnol. Biotechnol. Equip
                TBEQ
                tbeq20
                Biotechnology, Biotechnological Equipment
                Taylor & Francis
                1310-2818
                1314-3530
                4 March 2015
                17 February 2015
                : 29
                : 2
                : 205-220
                Affiliations
                [ a ]Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia , Skudai 81310, Johor, Malaysia
                [ b ]Department of Biotechnology and Medical Engineering, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia , Skudai 81310, Johor, Malaysia
                Author notes
                [* ]Corresponding author. Email: roswanira@ 123456kimia.fs.utm.my
                Article
                1008192
                10.1080/13102818.2015.1008192
                4434042
                26019635
                5c567b5b-cf94-45c6-a99c-6a4b1a40b912
                © 2015 The Author(s). Published by Taylor & Francis.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 August 2014
                : 7 October 2014
                Page count
                Figures: 1, Tables: 0, References: 192, Pages: 16
                Funding
                Funded by: Exploratory Research Grant Scheme from the Ministry of Higher Education Malaysia 10.13039/501100003093
                Award ID: ERGS R.J130000.7826.4L132
                Funded by: GUP scheme from the Universiti Teknologi Malaysia (UTM)
                Award ID: Q.J130000.2626.08J13
                This work was supported by the Exploratory Research Grant Scheme from the Ministry of Higher Education Malaysia [grant number ERGS R.J130000.7826.4L132] and the GUP scheme [grant number Q.J130000.2626.08J13] from the Universiti Teknologi Malaysia (UTM).
                Categories
                Review Articles; Agriculture and Environmental Biotechnology

                enzymes,immobilization,entrapment,surface analysis,nanoscale,atomic force spectroscopy,circular dichroism

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