Introduction

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The emergence of multidrug-resistant (MDR) bacteria is an extremely important threat to public health worldwide. The diversity of resistance mechanisms that contributes to the development of MDR may lead to pandrug resistance (PDR) [1,2]. Several international studies anticipate truly catastrophic scenarios on a global scale if effective solutions to tackle antimicrobial resistance are not rapidly found, with tens of million deaths per year and costs ascending to trillions of USD by 2050. Considering that the progress in discovering new antibiotics against MDR pathogens is very slow [3], it is conceivable that new strategies for controlling MDR or even PDR strains are urgently needed. This priority is currently being addressed by the use of multidisciplinary research efforts that aim to discovery new biomolecules able to effectively cope with the global emergence of MDR bacteria [4,5].

 

Classification

Enzybiotics consists of lytic enzymes that are naturally present in viruses, bacteria and in body fluids such as tears, saliva. They attract considerable interest as potential antibacterial tools [6]. These enzymes are responsible for digesting the bacterium cell wall. They are considered as excellent candidates for the development of novel therapeutics because they show a broad range of activity, are species-specific, possess high killing efficiency, are bactericidal (not just bacteriostatic), act in a short contact time, and they do not develop resistance [7,8,9]. Enzybiotics include lysins, bacteriocins, autolysins and lysoenzymens, which mainly belong to the class of peptidoglycan hydrolases as we mentioned above. Lysins are obtained from bacteriophages while autolysins are obtained from phage infected bacteria.

 

Bacteriocins (narrow spectrum antibiotics) are protein structure toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They are produced by non-pathogenic bacteria that normally colonize the human body. The loss of these harmless bacteria following antibiotic use may allow opportunistic pathogenic bacteria to invade the human body.

 

Lysins or endolysins are double stranded DNA bacteriophage encoded enzymes that slice the covalent bonds in peptidoglycan. These are basic enzymes having positive charge at pH lower than their isoelectric point, thus interact with the alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues.

 

Lysozymes, also known as muramidase or N-Acetylmuramide glycanhydrolase, damages the bacterial cell wall by catalyzing hydrolysis of 1,4 β-linkages between N-Acetyl D-glucosamine and N-Acetylmuramic acid residues in peptidoglycan. In human beings, lysozyme enzyme is encoded by LYZ gene. Lysozyme is regarded to be a natural antibiotic. It is a significant factor of innate immunity and a unique enzybiotic which exerts not only antibacterial activity but also antiviral, anti-inflammatory, anti-cancer and immunomodulatory activities.

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Autolysins are enzymes that hydrolyse the components of biological cell or tissue. Autolysins exists in all bacteria containing peptidoglycan. The peptidoglycan matrix is very rigid, thus these enzymes break down the matrix in to small sections so that growth and division of cell can occur. They act by hydrolyzing β (1,4) bond between N-acetylglucosamine and N-acetylmuramic acid.

 

Structure and Specificity

Peptidoglycan, also known as murein, is the major structural component of the bacterial cell wall. The peptidoglycan macromolecule forms a sacculus that surrounds the bacterial cytoplasmic membrane and confers the necessary mechanical resistance to avoid cell lysis as a result of turgor pressure. Therefore, uncontrolled breakdown of the murein structure typically results in osmotic cell lysis. The main structural features of peptidoglycan are linear glycan strands cross-linked by short peptides. The glycan strands are made up of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β-1→4 bonds. The terminal residues of the glycan strands are GlcNAc and 1,6-anhydroMurNAc, which is MurNAc with an intra-molecular ether-linkage from C-1 to C-6. These chains are interconnected by covalent crosslinks between short peptides on each MurNAc element. The D-lactoyl group of each MurNAc residue is substituted by a peptide stem whose composition is most often L-Ala-γ-D-Glu-meso-A2pm (or L-Lys)-D-Ala-D-Ala (A2pm, 2,6-diaminopimelic acid) in nascent peptidoglycan, the last D-Ala residue being lost in the mature macromolecule. Cross-linking of the glycan strands generally occurs between the carboxyl group of D-Ala at position 4 and the amino group of the diamino acid at position 3, either directly or through a short peptide bridge. Therefore, the chemical traits of this heteropolymer involve the presence of an unusual sugar (MurNAc), of γ-bonded D-Glu, of L–D (and even D–D) bonds and of nonprotein amino acids (e.g. A2pm). Thus, there are four enzyme classes that are classified as bacterial peptidoglycan hydrolases. Glycosidases, endopeptidases, amidohydrolases and lytic transglycosylases.  For example, the polysaccharide backbone can be possessed by glycosyl hydrolases (muramidases/lysozymes and glucosaminidases), the initial L-alanine of the pentapeptide stem can be cleavaged by alanine amidases, and the subsequent peptide bonds in the stem or cross bridge can be modified by endopeptidases [6,14].

 

LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Crystallography revealed a catalytic triad in the CHAPK active site consists of Cys54, His117 and Glu134. Similar structural and catalytic behaviour demonstrates streptococcal specific phage lysin PlyC. Moreover, group B streptococcal (GBS) bacteriophage lysin consists of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity targeting the alternating chain and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. The Acm domain of tbe GBS phage lysin possesses two acidic amino acid residues, Asp158 and Glul85, which are highly conserved in other proteins possessing the Acm domain.

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Application of Enzybiotics

Enzybiotics are used in many fields of daily life. They can be used in biomedice instead of commercial antibiotics or as specific antibiotic carriers into the targeted pathogenic bacterial cells. Additional potential applications of enzybiotics include several scientific areas such as agricultural (e.g., treatment of phytopathogens) [10], veterinary (e.g. treatment of animal pathogens) [11]. In addition, Enzybiotics can be used as food additives for controlling bacterial contamination in food industry (e.g. food-borne pathogens) [12] or as preservatives, in cosmetics and pharmaceutical products such as toothpastes, mouthwashes, aerosol sprays for respiratory infections, tear drops, ointments foe dermal and soft tissues.

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References

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