Features of the Hydrolysis Kinetics of Micrococcus lysodeikticus by Immobilized Lysozyme
Keywords:Lysozyme, Kinetics, Gelatin, Carboxymethyl cellulose, Polyvinyl alcohol cryogel
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.
Boddupalli BM, Mohammed ZN, Nath RA, Banji D. Mucoadhesive drug delivery system: an overview. J Adv Pharm Technol Res. 2010;1(4):381-7. DOI: 10.4103/0110-5558.76436
Mansuri S, Kesharwani P, Jain K, Tekade R, Jainae NK. Mucoadhesion: a promising approach in drug delivery system. React Funct Polym. 2016;100:151-72. DOI: 10.1016/j.reactfunctpolym.2016.01.011
Umesh K, Khuman L, Navneet P, Lekhraj, Jai P, Omkar, et al. Understanding the concept of mucoadhesive drug delivery system: a novel approach over conventional dosage forms. Res J Pharm Dosage Forms Technol. 2018;10(2):103-8. DOI: 10.5958/0975-4377.2018.00016.2
Asati S, Jain S, Choubey A. Bioadhesive or mucoadhesive drug delivery system: a potential alternative to conventional therapy. J Drug Delivery Therap. 2019;9(4-A):858-67. DOI: 10.22270/jddt.v9i4-A.3708
Dekina SS, Romanovska II, Ovsepyan AM, Molodaya AL, Pashkin II. Immobilization of lysozyme in polyvinyl alcohol cryogel. Biotechnologia Acta. 2014;7(3):69-73. DOI: 10.15407/biotech7.03.069
Dekina SS, Romanovska II, Leonenko II, Yegorova AV. Mucoadhesive gel with immobilized lysozyme: preparation and properties. Biotechnologia Acta. 2015;8(3):104-9. DOI: 10.15407/biotech8.03.104
Dekina S, Romanovska I, Ovsepyan А, Tkach F, Muratov E. Gelatin/carboxymethyl cellulose mucoadhesive films with lysozyme: development and characterization. Carbohydr Polym. 2016;147:208-15. DOI: 10.1016/j.carbpol.2016.04.006
Liburdu K, Benucci I, Palumbo F, Esti M. Lysozyme immobilized on chitosan beads: kinetic characterization and antimicrobial activity in white wines. Food Control. 2016;63:46-52. DOI: 10.1016/j.foodcont.2015.11.015
Yampol'skaya GP, Elenskii AA, Pan'kina NV, Tarasevich BN, Kulichikhin VG. Properties of carrageenan gels with immobilized lysozyme. Colloid J. 2009;71(2):271. DOI: 10.1134/S1061933X09020185
Shugar D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim Biophys Acta. 1952;8:302-9. DOI: 10.1016/0006-3002(52)90045-0
Cornish-Bowden E. Fundamentals of enzymatic kinetics. Moscow: Mir; 1979. 280 p.
Plokhinsky NA. Biometrics. Novosibirsk: Siberian Branch of the USSR Academy of Sciences; 1961. 364 p.
Linnik YV. The least squares method and the foundations of the mathematical-statistical theory of processing observations. Moscow: Fizmatgiz; 1962. 354 p.
Plesov AA, Berezin IV, Rabinovich ML. Kinetics of enzymatic reactions in heterogeneous systems. Bioorganicheskaya Khimiya. 1976;2(5):680-8.
Fersht E. Structure and mechanism of action of enzymes. Moscow: Mir; 1980. 432 p.
Krupyanko VI. Correction of equations for calculating the constants of two-parameter types of inhibition and activation of enzymes. Biochemistry. 2007;72(4):473-85.
Wilkinson GN. Statistical estimations in enzyme kinetics. Biochem J. 1961;80(2):324-32. DOI: 10.1042/bj0800324
Keleti T. Fundamentals of enzymatic kinetics. Moscow: Mir; 1990. 350 p.
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