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T4 lysozyme fused with cellulose-binding module for antimicrobial cellulosic wound dressing materials

Abouhmad, A. LU ; Mamo, G. LU ; Dishisha, T. LU ; Amin, M. A. and Hatti-Kaul, R. LU (2016) In Journal of Applied Microbiology 121(1). p.115-125
Abstract

Aims: Bacterial infection is a major challenge in wound care. Antimicrobial wound dressings are of great value for treating wound infections. Endolysins are evolving as a new class of antimicrobials with multiple applications. This study describes the production and evaluation of T4 lysozyme (T4Lyz), product of gene e of the T4 bacteriophage, fused with Cellulose Binding Module (CBM) for facile immobilization to cellulosic wound dressing. Methods and Results: Genes encoding T4Lyz-CBM and T4Lyz were cloned and expressed in Escherichia coli and the enzymes were purified by cation exchange chromatography. While the CBM tag did not alter the optimum pH and stability features of T4Lyz, the lytic activity of the fusion protein was lowered.... (More)

Aims: Bacterial infection is a major challenge in wound care. Antimicrobial wound dressings are of great value for treating wound infections. Endolysins are evolving as a new class of antimicrobials with multiple applications. This study describes the production and evaluation of T4 lysozyme (T4Lyz), product of gene e of the T4 bacteriophage, fused with Cellulose Binding Module (CBM) for facile immobilization to cellulosic wound dressing. Methods and Results: Genes encoding T4Lyz-CBM and T4Lyz were cloned and expressed in Escherichia coli and the enzymes were purified by cation exchange chromatography. While the CBM tag did not alter the optimum pH and stability features of T4Lyz, the lytic activity of the fusion protein was lowered. The bactericidal activity of T4Lyz-CBM, determined by viable count plating assay after 1 h incubation with Micrococcus lysodeikticus was 97·5% with 10 μg ml-1, and 99·96% and 95% for E. coli and Pseudomonas mendocina, respectively, with 200 μg ml-1 enzyme. T4Lyz-CBM was immobilized to wound dressing gauze with a capacity of 5·5 μg mg-1 matrix, whereas the unmodified T4Lyz did not exhibit any binding. The immobilized protein retained its bactericidal activity against Gram-positive and Gram-negative bacteria. Both free and immobilized T4Lyz-CBM, after heat denaturation, retained their bactericidal activities against Gram-negative bacteria only. The immobilized enzyme exhibited higher stability than the free enzyme when stored in dry form or in the presence of polyol stabilizers. Conclusion: Tagging T4Lyz with CBM provides a facile, irreversible binding to cellulosic wound dressing while retaining its activity. This approach may be suitable even for other antimicrobial enzymes and -peptides. Significance and Impact of the Study: The spread of antibiotic resistance requires innovative strategies for discovery and development of effective antimicrobial alternatives. This report presents a novel strategy for producing antimicrobial wound dressing materials.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antimicrobial activity, Cellulose binding module, Fusion protein, Immobilization, Storage stability, T4 lysozyme, Wound dressing
in
Journal of Applied Microbiology
volume
121
issue
1
pages
11 pages
publisher
Wiley-Blackwell
external identifiers
  • Scopus:84964671300
ISSN
1364-5072
DOI
10.1111/jam.13146
language
English
LU publication?
yes
id
9e70ee44-cc72-4d4f-a541-ed9bd6a9fadc
date added to LUP
2016-10-10 14:15:19
date last changed
2016-10-11 03:00:03
@misc{9e70ee44-cc72-4d4f-a541-ed9bd6a9fadc,
  abstract     = {<p>Aims: Bacterial infection is a major challenge in wound care. Antimicrobial wound dressings are of great value for treating wound infections. Endolysins are evolving as a new class of antimicrobials with multiple applications. This study describes the production and evaluation of T4 lysozyme (T4Lyz), product of gene e of the T4 bacteriophage, fused with Cellulose Binding Module (CBM) for facile immobilization to cellulosic wound dressing. Methods and Results: Genes encoding T4Lyz-CBM and T4Lyz were cloned and expressed in Escherichia coli and the enzymes were purified by cation exchange chromatography. While the CBM tag did not alter the optimum pH and stability features of T4Lyz, the lytic activity of the fusion protein was lowered. The bactericidal activity of T4Lyz-CBM, determined by viable count plating assay after 1 h incubation with Micrococcus lysodeikticus was 97·5% with 10 μg ml<sup>-1</sup>, and 99·96% and 95% for E. coli and Pseudomonas mendocina, respectively, with 200 μg ml<sup>-1</sup> enzyme. T4Lyz-CBM was immobilized to wound dressing gauze with a capacity of 5·5 μg mg<sup>-1</sup> matrix, whereas the unmodified T4Lyz did not exhibit any binding. The immobilized protein retained its bactericidal activity against Gram-positive and Gram-negative bacteria. Both free and immobilized T4Lyz-CBM, after heat denaturation, retained their bactericidal activities against Gram-negative bacteria only. The immobilized enzyme exhibited higher stability than the free enzyme when stored in dry form or in the presence of polyol stabilizers. Conclusion: Tagging T4Lyz with CBM provides a facile, irreversible binding to cellulosic wound dressing while retaining its activity. This approach may be suitable even for other antimicrobial enzymes and -peptides. Significance and Impact of the Study: The spread of antibiotic resistance requires innovative strategies for discovery and development of effective antimicrobial alternatives. This report presents a novel strategy for producing antimicrobial wound dressing materials.</p>},
  author       = {Abouhmad, A. and Mamo, G. and Dishisha, T. and Amin, M. A. and Hatti-Kaul, R.},
  issn         = {1364-5072},
  keyword      = {Antimicrobial activity,Cellulose binding module,Fusion protein,Immobilization,Storage stability,T4 lysozyme,Wound dressing},
  language     = {eng},
  month        = {04},
  number       = {1},
  pages        = {115--125},
  publisher    = {ARRAY(0xb40c010)},
  series       = {Journal of Applied Microbiology},
  title        = {T4 lysozyme fused with cellulose-binding module for antimicrobial cellulosic wound dressing materials},
  url          = {http://dx.doi.org/10.1111/jam.13146},
  volume       = {121},
  year         = {2016},
}