Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Abstract

A 20 fold purification of the gramicidin S synthesizing activity in a 11,OOOxg supernatant from Bacillus brevis has been achieved. The purification procedure involves precipitation of undesired material with streptomycin sulphate, followed by precipitation of gramicidin S synthesizing activity a t 40°/, (NH,),SO, saturation and fractionation on DEAE-Sephadex. All gramicidin S synthesizing activity appeared in one peak. It incorporated labelled phenylalanine, proline, valine, ornithine and leucine to the same extent into gramicidin S. The gramicidin S synthesizing activity was found to be stable in a 0.05 M potassium phosphate buffer (pH 7.1) containing glutathione (0.01 M) and glycerol (20°/,,, v/v) for a t least 3 days a t + 4" and four weeks at -20".Experiments with cell-free extracts from Bacillus brevis has established that gramicidin S is synthesized by a mechanism which is different from that of proteins [l-31. The next stage, naturally, is the isolation of the individual enzymes and the elucidation of the mechanism of synthesis. Recently, fractionation of the gramicidin S synthesizing activity from an 11,000~g supernatant of B. brevis was briefly described [4]. The present report is a more detailed account of and also an extension of these experiments. Since the gramicidin S synthesizing activity was found to be unstable, attempts were made to stabilize it. It was found that the gramicidin S synthesizing activity was stable a t $-4" in the presence of glycerol, glutathione (GSH) and phosphate buffer. Purification of the gramicidin S synthesizing activity from an 11,000 x g supernatant using streptomycin sulphate, (NH,),SO, and fractionation on a DEAE-Sephadex column resulted in a 20 fold purification with the activity in one peak. MATERIALS AND METHODS Growth of the OrganismThe Bacillus brevis strain ATCC 9999 was grown under aeration (5 1 airlmin) in 10 1 of the synthetic medium described by Eikhom, Jonsen, Laland

Activation of L‐ornithine by cell‐free extracts of Bacillus brevis catalyzing the synthesis of gramicidin S

“…Earlier studies on the biosynthesis of gramicidin S have indicated that RNA does not participate [ 1,3]. The present result is therefore in agreement with this earlier finding.…”

Synthesis of a linear gramicidin by a combination of biosynthetic and organic methods

“…The development of cell-free systems for the synthesis of GS1 and Ty made it possible to decipher the mechanism of biosynthesis of these cyclic decapeptides by nonribosomal systems (Saito et al, 1970;Kurahashi et al, 1969;Bredesen et al, 1968;Lipmann et al, 1971). These studies showed that the amino acids are activated by ATP on complementary enzymes of a molecular weight roughly proportional to the number of amino acids activated.…”

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis

“…This observation suggested auto-oxidation as an in vitro inactivation mechanism. Many reports of cellfree synthesis systems allude to the fact that reducing agents are required for short-term incubation (14)(15)(16)25) and long-term stabilization (2,7). Enhancement of stability by thiol reagents indicates that auto-oxidation (formation of enzyme disulfide) is compensated for by increased disulfide exchange with the thiol reagent (3,23).…”

“…Gramicidin S (GS) synthetase is an enzyme complex responsible for the nonribosomal biosynthesis of GS in Bacillus brevis. The complex includes both a heavy (280,000 daltons) and a light (100,000 daltons) enzyme and catalyzes the formation of the cyclic decapeptide GS: 2 In the first step of GS biosynthesis, the individual amino acids are activated by ATP, which results in the formation of aminoacyl adenylates (7,12,21). The light enzyme catalyzes activation and racemization of L-to Dphenylalanine, and the heavy enzyme activates I Presnt address: George Washington University Law School, Washington, DC 20003. the other constituent amino acids (10).…”