This work presents a method for purification of lysostaphin, a glycylglycine endopeptidase, from the culture liquid of S. simulans biovar staphylolyticus to homogeneity in a few steps. The method includes ultrafiltration and ion-exchange and hydrophobic chromatographies. The enzyme was isolated in preparative amounts with the yield of 51%. Some physical and chemical properties of the enzyme are described.
The rate of lysostaphin-catalyzed lysis of staphylococci follows the Michaelis-Menten equation at [E](0) << [S](0), i.e., the activity of the enzyme is proportional to its concentration. This equation is proposed for determining the specific activity of lysostaphin. The apparent activation energy of hydrolysis of pentaglycine bridges in Staphylococcus peptidoglycan is 77.9 kJ/mol.
Here, «host-vector» expression system of Brevibacillus choshinensis was developed and used for producing a recombinant lysostaphin with high-output. The recombinant plasmid pNCMO2/lsf12 was constructed, and its expression in Brevibacillus choshinensis (strain Brevibacillus choshinensis/pNCMO2/lsf12) provided a synthesis of the 27-kDa protein, which was secreted into the culture medium. Its specific staphylolitic activity being 557 U/mg at optimal pH (7.5-8.0) and temperature (50-55 °C) values was comparable with the natural and recombinant analogs. We hope that developed methods of a deep cultivation of the recombinant Brevibacillus choshinensis/pNCMO2/lsf12 strain for a high-yield production (up to 90 mg/L) and a single-stage purification of lysostaphin (up to 90% homogeneity) become the basis for the production of the enzyme on an industrial scale.
Brevibacillus choshinensis, ion-exchange chromatography, lysostaphin
The work was financially supported by the Grant No. 050 of Rospotrebnadzor «Monitoring of borreliosis pathogens circulation in regions of the Russian Federation and improvement of diagnostic tools for borreliosis»
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