Cloning And Expression Of The TSPpgh From Thermus Phage TSP4 And Its Activity Analysis

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria specially and efficiently after infection of host cell and releases of new phages.Phage lysine could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics.In this study,lysin with the name TSPgph coded by Themus phage TSP4 was studied.Gene of TSPgph was 501 bp,with GC content 57.09%.Homologous alignment analysis showed that TSPpgh has high homology with the peptidoglycan hydrolase from Thermus phage.Conserved domains search and phylogenetic tree analysis showed that TSPpgh belongs to peptidases M23 superfamily.Gene of TSPpgh was cloned to vector pET-28a to construct an expression plasmid pET28a-TSPpgh,and the plasmid was introduced into E.coli BL21 cells to obtain an express strain BL21/pET28a-TSPpgh.TSPpgh recombinant protein with molecular weight of 21 Kda was expressed in solubly in E.coli BL21 strain after induced by 1mM IPTG for 4h at 28℃.The inhibition effect of TSPpgh on different bacteria found that TSPpgh could obviously inhibit on growth of bacteria TC16,the host of TSP4,and inhibition effect on the thermophilic Bacillus sp.NC8 and E.col istrains were observed.TSPpgh had an enzyme activity range of 40 to 80℃ with optimum of 66.5℃,which was higher than most reported bacteriophage lytic enzyme.of the optimum pH value of TSPpgh was pH7-8.The effect of metal ions on enzyme activity showed that Na+ and Zn2+ had no effect on its activity,and it could inhibited by Mn2+ and Fe2+.on the contrary,its activity could stimulate by Mg2+.The main characteristic of TSPpgh could be summarized as low molecular weight,thermostability and inhibition growth of Gran-positive and negative cells.Based on structure entropy of TSPpgh,two mutants with lower structure entropy or higher structure entropy were designed and lower structure entropy mutant protein TSPpghl obtained.Comprason of the thermal stability of TSPpgh and TSPpgh 1 showed that TSPpgh1 thermal stability decreased.This result was consistent with theoretical of higher structure entropy,lower the thermal stability.This would benefit to explore the relationship between thermobility and structure entropy.