Enzymatic lysis of microbial cells Oriana Salazar Æ Juan A. Asenjo Abstract Cell wall lytic enzymes are valuable Bacteriolytic enzymes tools for the biotechnologist, with many applica- tions in medicine, the food industry, and agricul- Bacteriolytic enzymes have been greatly used in ture, and for recovering of intracellular products the biotechnology industry to break cells. Major from yeast or bacteria. The diversity of potential applications of these enzymes are related to the applications has conducted to the development of extraction of nucleic acids from susceptible lytic enzyme systems with specific characteristics, bacteria and spheroplasting for cell transforma- suitable for satisfying the requirements of each tion (Table 1). Other applications are based on particular application. Since the first time the lytic the antimicrobial properties of bacteriolytic enzyme of excellence, lysozyme, was discovered, enzymes. For instance, creation of transgenic many investigations have contributed to the cattle expressing lysostaphin in the milk gener- understanding of the action mechanisms and ated animals resistant to mastitis caused by other basic aspects of these interesting enzymes. streptococcal pathogens and Staphylococcus Today, recombinant production and protein engi- aureus (Donovan et al. 2005). Since this pepti- neering have improved and expanded the area of doglycan hydrolase also kills multiple human potential applications. In this review, some of the pathogens, it may prove useful as a highly recent advances in specific enzyme systems for selective, multipathogen-targeting antimicrobial bacteria and yeast cells rupture and other appli- agent that could potentially reduce the use of cations are examined. Emphasis is focused in broad-range antibiotics in fighting clinical infec- biotechnological aspects of these enzymes. tions. The use of lytic enzymes for the release of Keywords Bacteriolytic Á Cell lysis Á recombinant proteins from bacteria has been Intracellular protein recovering Á successful in many cases. For instance, Yang Yeast-lysing enzyme et al. (2000) used a temperature-sensitive lytic system for efficient recovery of recombinant proteins from Escherichia coli, and Zukaite and O. Salazar (&) Á J. A. Asenjo Biziulevicius (2000) applied lytic enzymes to Centre for Chemical Engineering and Biotechnology, accelerate the production of hyaluronidase by Department of Chemical Engineering and recombinant Clostridium perfringens in the Biotechnology, University of Chile, Beauchef 861, Santiago, Chile course of batch cultivation. e-mail: [email protected] Table 1 Present and Application References potential applications of microbial lytic enzymes Bacteriolytic enzymes DNA extraction from Gram-positive bacteria Niwa et al. (2005), Ezaki et al. (1990) Production of transgenic cattle resistant to Kerr and Wellnitz (2003), microbial infections Donovan et al. (2005) Antimicrobial for medical and Masschalck and Michiels (2003), food applications Loeffler et al. (2001), Fischetti (2003), Sava (1996) Release of recombinant proteins Zhang et al. (1999), Zukaite and Biziulevicius, (2000), de Ruyter et al. (1997) Yeast-lysing enzymes Preparation of protoplasts, cell fusion, and Kitamura (1982) transformation of yeast Production of intracellular enzymes Zomer et al. (1987) Pre-treatment to increase yeast digestibility Kobayashi et al. (1982) Preparation of soluble glucan polysaccharides Jamas et al. (1986) Alkali extraction of yeast proteins Kobayashi et al. (1982) Production of yeast extracts Conway et al. (2001) Food preservation Scott et al. (1987) Release of recombinant proteins from Asenjo et al. (1993) Saccharomyces cerevisiae Ferrer et al. (1996) Bacterial cell wall positive bacteria, but pre-treatment with a deter- gent (e.g. Triton X-100) or a cation chelating Based on the cell wall structure, bacteria are agent (as EDTA) is usually necessary to remove divided in Gram-positive and Gram-negative. the outer membrane of Gram-negative cells. Chemical composition and structure of the pep- tidoglycan in both types of bacteria are similar, Lysozyme, autolysins and endolysins though are much thinner in the Gram-negatives. The peptidoglycan layer, a polymer of N-acetyl-D- Enzymes that digest peptidoglycan of bacteria are glucosamine units b(1 fi 4)-linked to N-acety- collectively called murein hydrolases. Based on lmuramic acid, is responsible for the strength of their bond specificity, they are classified as: (i) the wall (Koch 1998). In Gram-positive bacteria, glycosidases, which split polysaccharide chains multiple layers of peptidoglycan are associated by (lysozymes or muramidases and glucosaminidases), a small group of amino acids and amino acid (ii) endopeptidases, which split polypeptide derivatives, forming the glycan-tetrapeptide, chains, and (iii) amidases, which cleave the which is repeated many times through the wall. junction between polysaccharides and peptides. Penta-glycine bridges connect tetrapeptides of Among muramidases, lysozyme is the best known adjacent polymers. Towards the out side, pepti- and best described; it is produced mostly from doglycan is connected to teichoic acids and hen egg white. Gram-negative bacteria, including polysaccharides. Gram-negative bacteria have a some major foodborne pathogens, are usually two-layer wall structure with a periplasmic space insensitive to lysozyme. In consequence, besides between them: an outer membrane composed of the use of Triton X-100 or EDTA, several other proteins, phospholipids, lipoproteins and lipo- strategies have been developed to expand the polysaccharides covers the inner, rigid peptido- uses of lysozyme to Gram-negative bacteria. glycan layer. These include: denaturation of lysozyme (Touch In Gram-negative bacteria, the outer mem- et al. 2003); modification of lysozyme by covalent brane precludes the access to lytic enzymes, attachment of polysaccharides (Aminlari et al. causing the major difference regarding to lytic 2005) or fatty acids (Ibrahim et al. 1991); attach- procedures. Lysozyme by itself can lyse Gram- ment of hydrophobic peptides to the C-terminal (Ibrahim et al. 1994) or cell permeabilization by autolysins and by autolysins from related high hydrostatic pressure (Masschalck et al. bacteria. 2001). Autolysins are ubiquitous enzymes but the best Extensive hydrolysis of peptidoglycan by lyso- characterized are those from Bacillus subtilis zyme results in cell lysis and death in a hypo (Smith et al. 2000), Staphylococcus aureus (Foster osmotic environment. Some lysozymes can kill 1995), and Streptococcus pneumoniae (Lopez bacteria by stimulating autolysin activity upon et al. 2000). Typically, autolysins have a modular interaction with the cell surface (Iacono et al. structure, with a N-terminal signal peptide fol- 1985). In addition, nonlytic bactericidal mecha- lowed by a second domain, which contains the nism, involving membrane damage without active site. In addition, these proteins harbor hydrolysis of peptidoglycan, has been reported repeat motifs flanking either the N-orC-terminal for c-type lysozymes, including human lysozyme of the catalytic domain. (Laible and Germaine 1985) and hen egg white Endolysins (or lysins) are lytic enzymes that lysozyme (HEWL) (Ibrahim et al. 2001; Mass- are functionally related to autolysins except they chalck et al. 2002). An increasing body of evi- are phage-encoded enzymes. They digest bacte- dence supports the existence of a nonenzymic rial peptidoglycan at the terminal stage of the and/or nonlytic mode of action of lysozyme phage reproduction cycle, allowing the release of (reviewed in Masschalck and Michiels 2003). the viral progeny out of the cell. Recent research Even if lysozymes from many different sources has not only revealed the diversity of these have been isolated and characterized, HEWL is hydrolases, but also yielded insights into their the only lysozyme that is currently used in most of modular organization and their three-dimensional the commercial applications. HEWL is produced structures (Fischetti 2005). Lysins can either be abundantly from their natural source, is inexpen- endo b-N-acetylglucosaminidase or N-acetylmu- sive and has a wider working range than several ramidase, endopeptidase or amidase. Most of the other lysozymes. endolysins are not translocated through the cyto- Efforts have been carried out for recombinant plasmic membrane to attack their substrate in the expression of lysozyme in E. coli; however, in this peptidoglycan; this action is controlled by holin, host HEWL is produced inactive as inclusion enzyme produced by the same phage lytic system, bodies (Schlo¨ rb et al. 2005). More successful have that disrupts the membrane and permits the been the attempts of expression in different access of the lytic enzyme to the peptidoglycan strains of Aspergillus niger (Archer et al. 1990; (Wang et al. 2000). Mainwaring et al. 1999; Gyamerah et al. 2002; Owing to their specificity and high activity, Gheshlaghi et al. 2005). endolysins have been employed for various in vitro HEWL is the most used lysozyme for bacterial and in vivo aims, such as food science, microbial cell wall disruption. However, some Gram-posi- diagnostic and for treatment of experimental tive bacteria are resistant to lysozyme. In those infections (Loessner 2005; Borysowski et al. cases, autolysins and endolysins can be applied. 2006). They are potentially useful antimicrobial Autolysins digest the cell wall peptidoglycan from agents for elimination of the opportunistic patho- cells that produce them