Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185 : I. Preparation of partially purified enzyme system and its properties

Abstract

1. A tyrocidine-synthesizing system was partially purified from the crude extract of Bacillus brevis by ammonium sulfate fractionation, protamine sulfate treatment followed by another ammonium sulfate fractionation to a specific activity of about 20 mμmoles of tyrocidine formation per h per mg of protein. The partially purified system absolutely required Mg 2+ (or Mn 2+), ATP and the tyrocidine constituent amino acids for its activity. The optimum conditions for tyrocidine synthesis were found to be 2 mM Mg 2+ (or Mn 2+), 4 mM ATP, and pH 7.8 in the presence of 0.2–0.4 mM constituent amino acids. 2. AMP was formed from ATP in the process of tyrocidine synthesis and 1 molecule of ATP was required for the formation of one peptide bond. 3. The partially purified system catalyzed ATP-PP i exchange reactions in the presence of glycine, l-alanine, l-lysine and l-isoleucine, besides 9 tyrocidine constituent l-amino acids. The other non-constituent amino acids of tyrocidine did not promote the exchange reaction. 4. Column chromatography on DEAE-cellulose separated the partially purified system into two distinct components, Components I and II, both of which were required for tyrocidine synthesis.