A Novel Method of Covalent Lysozyme Immobilization for the Development of Materials for Medical Applications

Covalently immobilized chemically modified lysozyme as a sorbent for bacterial endotoxins (lipopolysaccharides)

“…The reverse scheme (immobilization of the enzyme with its subsequent modification) is more complex from a technological point of view. The fact is that the pores of used by us matrix are rather large, therefore, lysozyme during immobilization is distributed inside the particles [3,6,20]. Modification of such a preparation with aldehydes in this case will be complicated by the diffusion of reagents inside the particles.…”

“…Relative bacteriolytic activity of immobilized modified lysozyme in comparison with immobilized unmodified lysozyme is higher against M. luteus cells (approximately one and a half times) and almost the same in the case of E. coli cells. As noted in our previous work in the case of immobilized lysozyme only a small part of the enzyme is sterically available to the bacterial cells substrate [3,6,20]. In general it can be said that the lysozyme amino groups involved in the modification and subsequent immobilization are not necessary to maintain bacteriolytic activity with respect to living bacterial cells.…”

“…Covalent immobilization of lysozyme was performed by attaching a protein to an aminated matrix using the approaches in our previous works [6,20]. For amination the matrix was first activated (oxidized) with NaIO 4 [21].…”

“…Endotixin adsorbtion is required for the extracorporeal treatment of sepsis [4,5]. In our recent work it was also shown that the new sorbent is blood-compatible so it can potentially be used in extracorporeal therapy technologies to remove pathogenic components from the patient’s bloodstream [3,6]. The practice of using extracorporeal therapy in the treatment of severe diseases including sepsis has significantly expanded in recent years [4,5,7].…”

“…In this work we set a goal to determine the resulting degree of modification (the number of modified amino groups per enzyme molecule) at various modifier/protein molar ratios in the reaction mixture, compare bacteriolytic properties of native and modified lysozyme, then obtain covalently immobilized lysozyme with different degrees of chemical modification and study bacteriolytic activity and sorption characteristics of new materials. As a polymer agarose matrix for lysozyme immobilization we chose WorkBeads 200SEC with a granule diameter of 200 μm for which its hemocompatibility and suitability for use in medicine in extracorporeal blood purification procedures was previously shown [3,6,14]. To determine the bacteriolytic activity we chose gram-negative Escherichia coli and gram-positive Micrococcus luteus cells as model bacteria substrates.…”