43] Gramicidin S synthetase

Abstract

Publisher Summary This chapter discusses the assay and purification procedure of gramicidin S synthetases. The following enzymic activities may be used for assaying gramicidin S synthetase: (1) synthesis of gramicidin S; (2) amino acid-dependent ATP- 32 PPi exchange; (3) amino acid-dependent ATP-[ 14 C] AMP exchange; (4) thioesterbonding of the individual amino acids, (5) ATP-dependent racemization of phenylalanine. Purification procedure involves cultivation of the microorganism, preparation of crude extract, streptomycin sulfate precipitation, ammonium sulfate precipitation, and chromatography on DEAE sephadex A-50, chromatography on sephadex G-200 alternative method or the separation of light and heavy enzyme: affinity chromatography. The biosynthesis of gramicidin S is the result of the concomitant functioning of a large number of catalytic activities. For instance, in the case of the heavy enzyme it has been estimated that about 18–20 different catalytic activities are involved. During the purification of gramicidin S synthetases that gives an almost homogeneous preparation of heavy enzyme, some 97% of its ability to synthesize the antibiotic is lost. The loss is particularly great during fractionation on the DEAE Sephadex G-50 and Sephadex G-200. Of the many catalytic activities involved in the biosynthesis, the catalytic activities responsible for amino acid activation are remarkably stable.