Abstract
Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His(6) enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His(6) enzymes showed highest activity at 37 °C. LysB-His(6) enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca(2+) and Mn(2+) enhanced the enzymatic activity of LysB-His(6) enzymes, while transition metal ions like Zn(2+) and Cu(2+) inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His(6) enzymes against mAGP complex was confirmed by LC-MS. LysB-His(6) enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.