Innovative Biosystems and Bioengineering (Mar 2020)
Features of the Hydrolysis Kinetics of Micrococcus lysodeikticus by Immobilized Lysozyme
Abstract
Background. The problem of the resistance of bacterial pathogens to antibiotics leads to a decrease in the effectiveness of therapy of infectious diseases, which in turn stimulates the search, modification or creation of new antibacterial agents, including lysozyme-polymer systems. The development of new biotechnological preparations of the enzyme with mucoadhesive properties, antimicrobial action, in the form of gels, hydrogel coatings or films, promising for use in medicine, is impossible without understanding the lysozyme functioning in an immobilized state. Therefore, studying the kinetic features of substrate hydrolysis by free and immobilized lysozyme, comparing the kinetic parameters of various forms of the enzyme is an urgent task. Objective. The aim of the paper is to study the kinetics of hydrolysis of Micrococcus lysodeikticus cells with lysozyme immobilized into gelatin, carboxymethyl cellulose sodium salt, and polyvinyl alcohol, as well as to compare kinetic characteristics with the parameters of the free enzyme. Methods. Immobilization of lysozyme into gelatin, sodium salt of carboxymethyl cellulose, polyvinyl alcohol was carried out by incorporation into a gel. The kinetic parameters of the functioning of the enzyme were determined by the initial lysis rates of M. lysodeikticus cells. The measurement results were analyzed using the Linuiver–Burke's and Haines' graphical methods, as well as the Cornish-Bowden–Eisenthal's computational method. Results. Kinetic features of the hydrolysis reaction of M. lysodeikticus cells by free and immobilized lysozyme were studied. The increase in Michaelis constant KM, the maximum reaction rate Vmax and the ratio KM/Vmax for the immobilized enzyme was shown. The tendency to increase the values of kinetic parameters is preserved both using the graphical linearization methods of Lineuiver–Burke and Haines and the Cornish-Bowden–Eisenthal's computational method, which is manifested in the transitional activation of lysozyme. In turn, a decrease in kcat/KM indicates a decrease in the rate of binding of the immobilized enzyme to the substrate. Conclusions. As a result of studying the kinetics of hydrolysis by three methods, it was shown that the action of the studied polymers (gelatin, sodium salt of carboxymethyl cellulose, and polyvinyl alcohol) is kinetically manifested in the transitional activation of the enzyme, according to the pattern of peptidoglycan of the cell wall of M. lysodeikticus. It has been revealed that the catalytic efficiency of lysozyme is most affected by the sodium salt of carboxymethyl cellulose, decreasing it by 37.8%, while gelatin and polyvinyl alcohol decrease this parameter by 31.8 and 18.8%, respectively. The results of the study are of fundamental and applied value, since they complement knowledge of the kinetic features of the functioning of immobilized forms of lysozyme, the interaction of the enzyme with polymers, and allow us to evaluate the catalytic efficiency of the obtained biotechnological products.
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