title: Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.
abstract: The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.
authors.affiliation: Department of Biochemistry and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand.
authors.forename: Nittaya
authors.initials: N
authors.lastname: Khakhum
authors.affiliation: Department of Microbiology and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand.
authors.forename: Umaporn
authors.initials: U
authors.lastname: Yordpratum
authors.affiliation: Department of Microbiology, Faculty of Science, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand.
authors.forename: Atcha
authors.initials: A
authors.lastname: Boonmee
authors.affiliation: Department of Microbiology and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand.
authors.forename: Unchalee
authors.initials: U
authors.lastname: Tattawasart
authors.affiliation: Department of Land, Air and Water Resources, University of California, Davis, 3308 Plant and Environmental Sci. Bld., Davis, CA, 95616, USA.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
authors.forename: Jorge L M
authors.initials: JLM
authors.lastname: Rodrigues
authors.affiliation: Department of Biochemistry and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand. rasana@kku.ac.th.
authors.forename: Rasana W
authors.initials: RW
authors.lastname: Sermswan
keywords: Endolysins; Enzyme; Gene expression; Peptidoglycan