Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes.

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-His6 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-His6 enzymes showed highest activity at 37 °C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 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.

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Optimization of Cellulase Production by Halobacillus sp. QLS 31 Isolated from Lake Qarun, Egypt.

A halophilic cellulase-producing bacterium was isolated from a sediment sample collected from Lake Qarun (Fayoum Province, Egypt). Molecular identification based on 16S rDNA amplification and sequencing revealed 99% homology with Halobacillus sp. and hence was designated as Halobacillus sp. QLS 31. Medium composition and culture conditions were optimized for enhancing the production of cellulase enzyme using the Plackett-Burman statistical design. Ten variables were evaluated for their influence on cellulase production. Carboxymethyl cellulose (CMC), zinc sulfate (ZnSO4), and inoculum size were found to exert a significant effect on cellulase productivity by Halobacillus sp. QLS 31. The maximum specific activity of cellulase enzyme was 48.08 U/mg. Following the predicted conditions, a 7.5-fold increase in cellulase specific activity (175.47 U/mg) was achieved compared to the basal medium (23.19 U/mg) under the following optimized conditions: temperature (30 °C), fermentation time (2 days ), pH value (9), CMC concentration (1%), inoculum size (1%), yeast extract concentration (0.1%), ammonium sulfate ((NH3)2SO4) concentration (0.1%), sodium chloride (NaCl) concentration (20%), and metal inducers: ZnSO4 (0.1%) and Ca/Mg ratio (0.01%). Thus, the results of this study provide an important basis for more efficient, cheap industrial cellulase production from halophilic Halobacillus sp. QLS 31.