Novel Highly Thermostable Endolysin from Thermus scotoductus MAT2119 Bacteriophage Ph2119 with Amino Acid Sequence Similarity to Eukaryotic Peptidoglycan Recognition Proteins

Abstract

ABSTRACTIn this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infectingThermusstrain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the speciesThermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with anMrof 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His30, Tyr58, His132, and Cys140) that form a Zn2+binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e.,T. scotoductus(100%),Thermus thermophilus(100%), andThermus flavus(99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e.,Escherichia coli(34%),Serratia marcescens(28%),Pseudomonas fluorescens(13%), andSalmonella entericaserovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception ofDeinococcus radiodurans(25%) andBacillus cereus(15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.