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.
Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr + or Gr − bacteria [1]. Their low toxicity as well as the absence of allergenicity and reactogenicity is confirmed by testing selected bacteriocins [2] [3]. Bacteriocins can be widely used as preservatives and antibiotic alternatives in medicine. Nisin, a Streptococcus lactis-derived bacteriocin, has been in practice in food industry for a long time. A relevant product contains about 2.5% of nisin. For medical use (e.g., when injected into the blood stream), highly purified drugs are required. However, the yield of bacteriocins accounts for no more than a few percents from the total activity in the culture liquid. In this paper, we propose methods (by example of two B. subtilis strains), allowing to increase the yield up to ~80%. It is believed that other bacteriocins may be purified by these methods and with the same yield.
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