N-15-LABELED AND C-13-LABELED MEDIA FROM ANABAENA SP FOR UNIVERSAL ISOTOPIC LABELING OF BACTERIOCINS - NMR RESONANCE ASSIGNMENTS OF LEUCOCIN-A FROM LEUCONOSTOC-GELIDUM AND NISIN-A FROM LACTOCOCCUS-LACTIS

A procedure for universal C-13 and/or N-15 labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [C-13]bicarbonate and/or sodium [N-15]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both C-13 and N-15 remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [C-13, N-15]nisin A using H{C-13}MQC, H{C-13}MBC, 2D INADEQUATE, and H{N-15}MQC techniques confirmed H-1 spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform C-13 labeling occurs unless the nutrient source is completely isotopically enriched at high level (greater-than-or-equal-to 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H{C-13}MQC to unlabeled and [C-13] leucocin A afforded the complete H-1 and C-13 assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.