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Lytic enzyme from lysates of Streptomyces venezuelae infected with actinophage MSP2
Abstract:
A lytic enzyme was purified 600-fold with 12% recovery from lysates of Streptomyces venezuelae S13 infected with actinophage MSP2. The purified enzyme preparation was homogeneous as shown by polyacrylamide electrophoresis. The enzyme was active over a pH range 6.0 to 9.0 with a maximum at pH 7.5. The pH profile for stability was sharp, with an optimum at pH 7.5. Maximal activity occurred between 30 and 35 C. The enzyme was stable at 20 C or less. A 30-min exposure to 25, 30, 35, 40, 45, and 50 C produced an inactivation of 3, 40, 77, 82, 93, and 100%, respectively. Lytic activity was stimulated fivefold by either 5 x 10(-3)m Mg(2+) or Mn(2+) and three- and twofold by Ca(2+) and Ba(2+), respectively. Addition of Na(+), K(+), NH(4) (+), or Li(+) to the tris(hydroxymethyl)aminomethane-hydrochloride buffer did not alter the rate of lysis. Enzyme activity was inhibited 74 and 27% by 10(-4) and 10(-5)m ethylenediaminetetraacetic acid (EDTA), respectively. The inhibition by EDTA was reversed partially by addition of Mg(2+). Lytic activity was abolished by either 5 x 10(-4)m HgCl(2) or p-hydroxymercuribenzoate, whereas 5 x 10(-4)m CuSO(4) inhibited 72%. Cell wall solubilization paralleled the release of N-terminal amino groups and reached a level of 0.23 mumole per mg of cell walls. No release of reducing power was detected in treated or untreated cell wall suspensions. Tests for proteolytic activity were negative.
Insights
A novel lytic enzyme from Streptomyces venezuelae S13 was purified and characterized. This enzyme effectively degrades bacterial cell walls, showing optimal activity at pH 7.5 and moderate temperatures, and is influenced by specific metal ions.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Actinophages are viruses that infect actinomycetes, such as Streptomyces venezuelae.
- Lytic enzymes produced by phages or bacteria play a crucial role in cell wall degradation.
- Understanding these enzymes is vital for applications in biotechnology and antimicrobial strategies.
Purpose of the Study:
- To purify and characterize a lytic enzyme from Streptomyces venezuelae S13 infected with actinophage MSP2.
- To determine the optimal conditions for enzyme activity and stability.
- To investigate the effects of various metal ions and inhibitors on enzyme function.
Main Methods:
- Purification of the lytic enzyme using standard biochemical techniques, achieving 600-fold purification.
- Characterization of enzyme activity across a range of pH and temperatures.
- Assessment of enzyme stability and the impact of divalent cations (Mg2+, Mn2+, Ca2+, Ba2+) and inhibitors (EDTA, HgCl2, p-hydroxymercuribenzoate, CuSO4).
Main Results:
- The purified enzyme was homogeneous and active between pH 6.0-9.0 (optimum 7.5).
- Optimal activity was observed between 30-35°C; the enzyme was stable at 20°C or below.
- Lytic activity was significantly enhanced by Mg2+ or Mn2+ and inhibited by EDTA and heavy metal ions. Cell wall solubilization correlated with N-terminal amino group release.
Conclusions:
- A potent lytic enzyme was successfully isolated and characterized from Streptomyces venezuelae S13.
- The enzyme's properties suggest potential applications in cell wall lysis and microbial control.
- Further research could explore its specific substrate targets and potential in biotechnological processes.