Category:Team EKSUemerald

The predicted endolysin (ADE34958) of phage KP15 is very similar to the endolysin (AEX26632) of phage KP27.

The only difference is only in one amino acid (glutamic acid of KP15 endolysin is replaced by glutamine in the KP27 endolysin in the seventeenth position from N-terminus) (results section, subsection;

Description of KP15 and KP27 lysis system).  

Figure 4 AND S4 show the digestion of E. coli peptidoglycan sacculus KP27 endolysin and shows that KP27 endolysin is a l-alanyl-d-glutamate endopeptidase. It further shows that KP27 endolysins has an endopeptidase activity cleaving between l-Ala and d-Glu of the stem peptide and no d-alanyl-d-alanine carboxypeptidase activity.

Figure 4 AND S4 show the digestion of E. coli peptidoglycan sacculus KP27 endolysin and shows

that KP27 endolysin is a l-alanyl-d-glutamate endopeptidase. It further shows that KP27 endolysins has an endopeptidase activity cleaving between l-Ala and d-Glu of the stem peptide and no d-alanyl-d-alanine carboxypeptidase activity.

Figures 3, 4 and 5 show PlyPalA has lytic activity on P. larvae cells.

Table 1 showed that PlyPalA had lytic activity against different P. larvae genotypes and the lytic activity varied from genotype-to-genotype. ERIC genotype I members were the most susceptible to PlyPalA lytic activity

Figure 1 and 3 show that purified proteins from all C-terminal (Cell Wall Binding [CBD])

- Green Flourescent Protein (GFP)fusion constructs, had evident binding activity. In all cases, no binding activity of unfused GFP, used as a negative control, was detected.

Figure 6 shows that LysPBC1 is a putative N-acetylmuramoyl-L-alanine amidase consisting of

an N-terminal type 3 amidase domain (PF01520) and a C-terminal amidase02_C domain (PF12123). Table 1 shows that when purified LysPBC1 was added exogenously it lysed all strains of B. cereus group bacteria, including B. cereus, B. thuringiensis, B. mycoides, and B. weihenstephanensis,

The authors claim that 'BlastP, Pfam and HHpred identified an N-acetylmuramoyl-L-alanine

amidase belonging to the Amidase_2 family (Pfam 01510) at the N-terminus encompassing two third of the protein. CDD database identified 5 putative amidase catalytic residues: two histidines (position 29 and 129), a phenylalanine (position 53), a lysine (position 135) and a cysteine (position 137)' (First paragraph of the results section).

Table 2 however confirmed that PlyPl23 has lytic activity and also shows the lytic spectrum of PlyPl23

when assessed against a panel of 20 P. larvae isolates and 4 additional strains belonging

to other related bacterial species (Bacillus cereus, Lactobacillus paracasei, 

Lactobacillus pentosus, Bacillus subtilis), and compared with that of the respective phage phiIBB_Pl23. Results showed that while the phage phiIBB_Pl23 was able to lyse 16 of the 20 strains (80%), its endolysin (PlyPl23) was active against all the tested Paenibacillus larvae strains. The Bacillus and Lactobacillus strains were not lysed by the phage or by the enzyme

FIGURE 6 shows that PlyCA contains a functional Cysteine, Histidine-Dependent Amidohydrolase Peptidase

(CHAP) Domain confirming it to have amidase activity, because it is able to cleave the amide bond between N-acetyl muramic acid and L-alanine in the streptococcal peptidoglycan (Fischetti et al., 1972).

Figure 1 shows that PlyAB1 (R4IQ32) is a homologue of LysABP-01 (accession no: W0M2E9) and

LysAB2 (accession no: F1BCP4) whose endolysin activity have been demonstrated in PMID:27656173 [LysABP-01 (accession no: W0M2E9)] and PMID:21264466 [LysAB2 (accession no: F1BCP4)]

Figure 3 shows antibacterial (lytic) activity of LysABP-01

Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys-2

on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.

Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys

on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. the HPLC profile reveal the hydrolytic specificities for Lc-Lys. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys has an N-acetylmuramoyl-L-alanine amidase specificity,

Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys-2

on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.

Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys

on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. the HPLC profile reveal the hydrolytic specificities for Lc-Lys. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys has an N-acetylmuramoyl-L-alanine amidase specificity,

fig. 3B and Table 3 show the hydrolytic specificity of the recombinant pure Lc-Lys-2 on Lactobacillus casei-purified Peptidoglycan (PG). The PG fragment obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2.

MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.