Orientation of Spin-Labeled Lysozyme from Chicken Egg White Immobilized on Porous Oxide Carriers
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Applied Magnetic Resonance
ORIGINAL PAPER
Orientation of Spin‑Labeled Lysozyme from Chicken Egg White Immobilized on Porous Oxide Carriers Denis O. Antonov1 · Natalia A. Chumakova2 · Elena G. Kovaleva1 Received: 30 March 2020 / Revised: 12 June 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract The spectroscopy of electron paramagnetic resonance, unlike optical methods, allows observing paramagnetic molecules inside a non-paramagnetic matrix of any morphology, which makes it possible to investigate the behavior of proteins immobilized on porous carriers. In the present work the preferential orientation of lysozyme from chicken egg white, spin-labeled in position his-15, immobilized on the surface of a series of porous oxide carriers, has been defined by computer simulation of the dynamic EPR spectra.
1 Introduction The immobilization of enzymes on a solid carriers is important for their industrial, medical and food applications since it allows to reduce cost by reusing heterogeneous enzymatic catalysts [1, 2], as well as to minimize protein contamination of the substrate. In some cases, immobilization can increase the stability of enzymes to thermal denaturation or to presence of organic solvents [3]. To date, many methods of surface immobilization of proteins have been developed, such as physical adsorption when the enzyme is attached to the carrier due to opposite charges on the surface of the protein and on the surface of the carrier [4], encapsulation of proteins into the porous glasses matrixes using sol–gel technology [5], and immobilization of the enzyme through the formation of covalent bonds of the protein with functional groups on the surface of the carrier [6]. There are many strategies for covalent binding of the enzyme to the carrier both using the intrinsic reaction groups on the surface of the protein and by means of directed mutagenesis to add the necessary groups at certain positions, which makes it possible to orient the enzyme on the surface of the carrier in the manner necessary for use [7]. The most widely used * Denis O. Antonov [email protected] 1
Ural Federal University Named After the First President of Russia B.N.Yeltsin, Yekaterinburg, Russia
2
Moscow State University Named After M.V. Lomonosov, Moscow, Russia
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method is the covalent binding by reactions between epoxy groups on the surface of the carrier and amino groups on the surface of the protein [6, 8]. As well the protein attachment to the surface via glutaraldehyde is often used. The latter reacts with amino groups both on the surface of the protein and on the surface of the carrier, thereby providing covalent crosslinking [1, 6, 18]. The immobilization of the enzyme on the surface of the carrier is known to result in partial loss of catalytic activity. It can be caused by steric hindrances, i.e. lack of access to the catalytic center of the protein, or its partial denaturation. Catalytic methods do not allow separating these factors. The techniqu
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