Production and Purification of the Thermophilic Bacteriophage TP-84

Abstract

A new procedure for production and purification of the thermophilic bacteriophage TP-84 in high yields is described. Cultures of Bacillus stearothermophilus strain 10, enriched with nutrients to obtain heavy growth and to prevent sporulation and maintained at a pH of 6.5, were infected with the phage in a 100-liter fermentor. Addition of magnesium chloride (0.01 M) and a temperature of 58 C were essential for maximal phage production. Phage (5 × 10 11 infective particles/ml) was precipitated with polyethylene glycol (molecular weight 6,000) in the presence of sodium chloride and was further purified by cesium chloride density centrifugation.

Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent

“…E. coli BL21(DE3) (B F − ompT hsdS(r B − m B − ) dcm + Tet r galλ (DE3) endA Hte), E. coli BL21(DE3)pLysS (B F − ompT hsdS(r B − m B − ) dcm + Tet r galλ (DE3) endA Hte pLysS), and E. coli ArcticExpress(DE3) (B F − ompT hsdS(r B − m B − ) dcm + Tet r galλ (DE3) endA Hte cpn10 cpn60 Gent’) (Agilent Technologies, Palo Alto, CA, USA) were used for gene expression. Bacillus stearothermophilus / G. stearothermophilus strain 10 host for bacteriophage TP-84 was sourced from the collection of Prof. Piotr Skowron and is identical to the host strain published previously [ 2 , 3 ]. Geobacillus ICI was isolated by Prof. Skowron’s team from hot springs in Iceland.…”

“…Furthermore, the graph in Figure 5 shows that the heat denaturation of recombinant TP84_28 endolysin is irreversible and the characteristic shape of the graph at 90 °C indicates the aggregation of the protein molecules. These thermostability assay results, obtained by three independent methods, indicate that the recombinant enzyme expressed in the mesophile retains the characteristics of the native enzyme and the upper activity and stability limit correspond with the highest temperature that can support a G. stearothermophilus /TP-84 system [ 3 ].…”

“…The lytic cycle of TP-84 lasts 22–24 min under optimal conditions: (i) propagation at a 55–58 °C temperature, (ii) media supplemented with glucose/fructose, and (iii) the presence of Ca 2+ ions [ 2 , 3 ]. Remarkably, it can grow through an enormous temperature range of 30–80 °C [ 4 ].…”

Bacteriophages of Thermophilic ‘Bacillus Group’ Bacteria—A Review

“…10 12 . However, omitting the sugar addition decreased the titer approximately 1000-fold [ 39 ], while omitting divalent cations had a dramatic effect on final TP-84 yields, causing an approximate 200-fold decrease in titer [ 41 ]. The temperature growth range of 43–76 °C (optimal 55–60 °C) matched that of the hosts growth range.…”

“…The latent period was 22–24 min. Clear plaques were surrounded by a turbid ‘halo’ ([ 41 ] Lubkowska et al, unpublished data), indicating diffusion of an enzyme, possibly glycosylase-depolymerase, degrading the bacterial envelopes. The bacteriophage has a hexagonal head, 53 nm in diameter with a 131 nm long 3–5 nm wide tail.…”