Peptidoglycan Hydrolytic Activity of Bacteriophage Lytic Proteins in Zymogram Analysis

Rodríguez-Rubio, Lorena; Donovan, David M.; Martínez, Beatriz; Rodrı́guez, Ana; Garcı́a, Pilar

doi:10.1007/978-1-4939-8940-9_9

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…The enzymatic activity of the protein was determined through zymographic assay [ 34 ]. Purified TP84_28 endolysin was separated by electrophoresis using 12% SDS-PAGE gel that included the G. stearothermophilus strain 10 host cells.…”

Section: Methodsmentioning

confidence: 99%

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

Żebrowska

Żołnierkiewicz

Ponikowska

et al. 2022

IJMS

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The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.

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Section: Methodsmentioning

confidence: 99%

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

Żebrowska

Żołnierkiewicz

Ponikowska

et al. 2022

IJMS

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“…These results are consistent with Rodri ́guez-Rubio et al who reported that the binding properties of cell wall-binding domain influence the lytic activity of catalytic modules. 36 The high activity of the CD L −BD S (3:1) construct against S. aureus strain (ATCC 33807) was intriguing and led us to test this construct further by comparing to the C-terminalbiotinylated Lst−SA and free Lst against a second S. aureus strain (ATCC 6538) and an S. epidermidis strain (ATCC 12228). The CD L −BD S (3:1) construct was highly active against S. aureus 33807 with an observed >5 log reduction.…”

Section: Resultsmentioning

confidence: 99%

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity

et al. 2019

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Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL–BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

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“…There are many circumstances when the presence, the purity or even the quantity of a protein in a solution is evaluated by polyacrylamide gel electrophoresis (PAGE) [1][2][3]. When the analyzed protein is an enzyme, its activity can be estimated combining electrophoretic separation with zymogram [4].…”

Section: Introductionmentioning

confidence: 99%

A fast and sensitive zymography method of peroxidase activity determination using sodium acetate buffer

Menghiu¹,

Iancu²,

Ostafe³

2019

Studia UBB Chemia

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By changing the buffer composition from phosphate to acetate (in both cases, the pH value was 6.0), the sensitivity of the estimation of peroxidase activity in polyacrylamide gels was improved from 4 µg/well to 0.4 µg/well. The optimized method, including electrophoretic separation and zymogram technique realized with guaiacol as substrate, can be performed in only 135 minutes. The improved method was applied to monitor the expression of recombinant peroxidase in a Gram-negative bacterium: the peroxidase activity was detected at 4 h after induction of expression.

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Peptidoglycan Hydrolytic Activity of Bacteriophage Lytic Proteins in Zymogram Analysis

Cited by 4 publications

References 27 publications

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

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

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity

A fast and sensitive zymography method of peroxidase activity determination using sodium acetate buffer

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