|
Cloning, expression, purification and investigation of activity of Klebsiella pneumoniae phage KP27 endolysin
|
Zeynab Zare   , Mehdi Imani *  , Safiyeh Aghazadeh |
| Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran |
|
|
Abstract: (415 Views) |
Background and Aim: Endolysin of phage KP27 is a protein with a molecular mass of 14.7 kDa, which has endopeptidase activity against bacterial cell walls. Due to limited research on endolysin KP27 and its superiority against harsh environmental conditions such as temperature and pH, the aim of this study is recombinant production and investigation of its antibacterial activity against two gram-negative and positive bacteria
Methods: KP27 endolysin gene was cloned between two restriction enzymes NcoI and XhoI in plasmid pET-28a(+). E. coli BL21 (DE3) was used for expression and production and Purification was performed by Ni2+-NTA sepharose chromatography. Bactericidal activity tests, MIC and MBC against Escherichia coli and Staphylococcus aureus bacteria and cell wall degradation were used to investigate the antibacterial and enzymatic activities of endolysin, respectively. Statistical analysis and generation of graphs were achieved by Graphpad Prism software.
Results: Endolysin KP27 was successfully cloned and produced with high efficiency in the amount of 28.5 mg/L of culture medium. Bactericidal activity against Escherichia coli and Staphylococcus aureus bacteria was over 50% at the concentrations of 4 µg/ml. For Escherichia coli, MIC and MBC were calculated 16 and 64 µg/ml while for Staphylococcus aureus they were 8 and 32 µg/ml, respectively. Evaluating the cell wall degradation activity of endolysin indicated a 20% decrease in the turbidity of the solution after half an hour incubation with endolysin.
Conclusion: Considering the high expression and production as well as its high activity against both Gram-negative and Gram-positive bacteria, endolysin KP27 can be suggested as a convenient candidate for further applied research and subsequent industrialization instead of antibiotics.
|
|
| Keywords: Endolysin KP27, Bacteriophage, Recombinant protein, Cell wall, Antibiotic resistance |
|
|
Full-Text [PDF 1015 kb]
(180 Downloads)
|
|
Type of Study: Original Research |
Subject:
Others...
|
|
|
|
|
|
|
| Add your comments about this article |
|
|
|
