Food Sci. Biotechnol. 23(1): 151-155 (2014) DOI 10.1007/s10068-014-0020-9 Kinetic Study of a Hen-Egg White Lysozyme-based Oenological Preparation Esti Marco, Liburdi Katia, Palumbo Fabio, Benucci Ilaria, and Garzillo Anna Maria Vittoria Received: 12 March 2013 / Revised: 16 July 2013 / Accepted: 24 July 2013 / Published Online: 28 February 2014 © KoSFoST and Springer 2014 Abstract The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates i) glycol chitosan, as a non-cellular synthetic substrate, and ii) Oenococcus oeni , as a microbial substrate for muramidase activity, was investigated in wine-like acidic medium (tartaric buffer, pH 3.2). The good reproducibility and reliability of results revealed that both substrates are useful for lysozyme kinetics studies. Both the chitinolytic and muramidase activity of lysozyme were affected by uncompetitive substrate inhibition. However, the O. oeni effect occurred at very high concentration with respect to the typical wine bacterial content, even when microbial population reaches its maximum growth level during malolactic fermentation. The optimum pH and temperature for lysozyme activity were very far from the usual wine handling conditions, but residual enzyme activity was observed at pH 3.2 and 20 o C, confirming that the oenological application of lysozyme is a useful practice. Keywords: lysozyme, muramidase activity, chitinolytic activity, kinetic study, wine-like acidic medium Introduction Lysozyme (E.C. 3.2.1.17), also known as muramidase or N-acetylmuramide-glycanhydrolase, typically extracted from hen egg white, has been widely used in the wine industry to control Gram-positive bacteria growth and malolactic fermentation (MLF) since the 1990s. Hen’s egg white lysozyme (HEWL) has an isoelectric point of 10.5-11.0 and a molecular mass of approximately 14,500 Da. It acts by splitting the beta-(1-4) linkage between N-acetylmuramic acid (NAM) and N-acetyl-glucosamine (NAG), two components of the peptidoglycan (synonyms: murein, mucopeptide, glycosaminopeptide) that makes up the Gram-positive bacterial cell wall. Thus, lysozyme treatment leads to lysis and death in susceptible bacteria. The ability of lysozyme to reduce lactic bacterial flora in buffer solution has been investigated (1). The most commonly used method of measuring HEWL bacteriolytic action utilises Micrococcus luteus as cell substrate (2). Oenococcus oeni , an oenological strain recognised as the major bacterium involved in malolactic fermentation during the winemaking process, was employed as a HEWL substrate (3). Because murein is insoluble, the catalytic mechanism of lysozyme was also studied using a synthetic substrate, a hexasaccharide of N-acetylglucosamine, which has been reported as covalent glycosyl-enzyme intermediate during the HEWL catalytic cycle (4). Most lysozymes exhibit chitinase activity in addition to muramidase activity, most likely as a result of the similarity between peptidoglycan, the natural substrate of HEWL, and chitin, a homopolymer of beta-1,4-linked N-acetyl glucosamine (5). However, little is known about the kinetic parameters of lysozyme in wine-like acidic medium. Therefore, the present work characterised the beta 1-4 hydrolytic activity of a HEWL oenological preparation in tartaric acid buffer (pH 3.2). Enzymatic activity toward specific substrates was tested: i) glycol chitosan, a low molecular weight homopolymer of beta-1,4-linked N-acetyl glucosamine, as non-cellular synthetic substrate to test chytinolitic activity, and ii) O. Esti Marco, Liburdi Katia (), Palumbo Fabio, Benucci Ilaria Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Tuscia University, via S. Camillo de Lellis, 01100 Viterbo, Italy Tel:+39-0761-357426; Fax:+39-0761-357498 E-mail: k.liburdi@unitus.it Garzillo Anna Maria Vittoria Department of Ecological and Biological Sciences, Tuscia University, Largo dell’Universit, 01100 Viterbo, Italy RESEARCH ARTICLE