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Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

Farris MH, Steinberg AD - Lett. Appl. Microbiol. (2014)

Bottom Line: Mitrecin A consists of 127 amino acids arranged in modular domains of activity.It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases.The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia.

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Affiliation: Department of Advanced Technology, The MITRE Corporation, McLean, VA, USA.

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Bacteriolytic activity of Mitrecin A. Residual viability of the Yersinia pseudotuberculosis indicator culture (a) was measured by determining the colony-forming units per millilitre after 16 h exposures of the culture to various concentrations of Mitrecin A. The regression coefficient (R2) of the plot is for the best-fit third-order polynomial. Microslide assays were performed to rapidly visualize the enzymatic activity of Mitrecin A against susceptible bacteria. In an illustration of the microslide assay (b), enzymatic activity for Mitrecin A (well 1, 1 μg), bovine serum albumin (well 2, 1 μg) and lysozyme (well 3, 1 μg) is shown against Aeromonas hydrophila.
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fig04: Bacteriolytic activity of Mitrecin A. Residual viability of the Yersinia pseudotuberculosis indicator culture (a) was measured by determining the colony-forming units per millilitre after 16 h exposures of the culture to various concentrations of Mitrecin A. The regression coefficient (R2) of the plot is for the best-fit third-order polynomial. Microslide assays were performed to rapidly visualize the enzymatic activity of Mitrecin A against susceptible bacteria. In an illustration of the microslide assay (b), enzymatic activity for Mitrecin A (well 1, 1 μg), bovine serum albumin (well 2, 1 μg) and lysozyme (well 3, 1 μg) is shown against Aeromonas hydrophila.

Mentions: Optimal catalytic activities for Mitrecin A were achieved at 26°C in solution of pH 9 with 1% saline (Fig. 3). The enzyme is sensitive to acidic conditions below pH 6 and shows complete inactivation at temperatures above 55°C. In considering potential downstream applications of the enzyme, pH 7·4 was held constant under varying saline (Fig. 3b) and temperature (Fig. 3c) treatments to illustrate the activity of Mitrecin A at human physiological pH. The bacteriolytic effects of Mitrecin A indicate a resistant population within the bacterial target population, resulting in turbid zones of lysis in microslide assays and a lack of broth clearing during viability bacteriolytic assays. For example, in the kill curve viability assay (Fig. 4a), increased concentrations of Mitrecin A above 0·15 mg ml−1 returned diminished kill results. Similar subpopulation susceptibility to endolysins of actinomycete bacteriophage has been previously described (Higgins and Lechevalier 1969).


Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

Farris MH, Steinberg AD - Lett. Appl. Microbiol. (2014)

Bacteriolytic activity of Mitrecin A. Residual viability of the Yersinia pseudotuberculosis indicator culture (a) was measured by determining the colony-forming units per millilitre after 16 h exposures of the culture to various concentrations of Mitrecin A. The regression coefficient (R2) of the plot is for the best-fit third-order polynomial. Microslide assays were performed to rapidly visualize the enzymatic activity of Mitrecin A against susceptible bacteria. In an illustration of the microslide assay (b), enzymatic activity for Mitrecin A (well 1, 1 μg), bovine serum albumin (well 2, 1 μg) and lysozyme (well 3, 1 μg) is shown against Aeromonas hydrophila.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4238840&req=5

fig04: Bacteriolytic activity of Mitrecin A. Residual viability of the Yersinia pseudotuberculosis indicator culture (a) was measured by determining the colony-forming units per millilitre after 16 h exposures of the culture to various concentrations of Mitrecin A. The regression coefficient (R2) of the plot is for the best-fit third-order polynomial. Microslide assays were performed to rapidly visualize the enzymatic activity of Mitrecin A against susceptible bacteria. In an illustration of the microslide assay (b), enzymatic activity for Mitrecin A (well 1, 1 μg), bovine serum albumin (well 2, 1 μg) and lysozyme (well 3, 1 μg) is shown against Aeromonas hydrophila.
Mentions: Optimal catalytic activities for Mitrecin A were achieved at 26°C in solution of pH 9 with 1% saline (Fig. 3). The enzyme is sensitive to acidic conditions below pH 6 and shows complete inactivation at temperatures above 55°C. In considering potential downstream applications of the enzyme, pH 7·4 was held constant under varying saline (Fig. 3b) and temperature (Fig. 3c) treatments to illustrate the activity of Mitrecin A at human physiological pH. The bacteriolytic effects of Mitrecin A indicate a resistant population within the bacterial target population, resulting in turbid zones of lysis in microslide assays and a lack of broth clearing during viability bacteriolytic assays. For example, in the kill curve viability assay (Fig. 4a), increased concentrations of Mitrecin A above 0·15 mg ml−1 returned diminished kill results. Similar subpopulation susceptibility to endolysins of actinomycete bacteriophage has been previously described (Higgins and Lechevalier 1969).

Bottom Line: Mitrecin A consists of 127 amino acids arranged in modular domains of activity.It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases.The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Technology, The MITRE Corporation, McLean, VA, USA.

Show MeSH
Related in: MedlinePlus