title: Scalable purification of the lantibiotic nisin and isolation of chemical/enzymatic cleavage fragments suitable for semi-synthesis.
abstract: Herein, we describe a scalable purification of the lantibiotic nisin via an extraction/precipitation approach using a biphasic system, which can be carried out up to 40-80 gram scale. This approach results in an at least tenfold enrichment of commercially available preparations of nisin, which usually contain only 2.5% of the desired peptide, to allow further purification by preparative HPLC. As a follow-up study, the enriched nisin sample was digested either by trypsin or chymotrypsin, or treated by CNBr, and these reactions were monitored by LC-MS to identify and characterize the obtained fragments. Two previously unknown cleavage sites have been identified: Asn20-Met21 and Met21-Lys22 for trypsin and chymotrypsin, respectively. Furthermore, a novel and convenient enzymatic approach to isolate the native nisin C-ring [nisin fragment (13-20)] was uncovered. Finally, by means of preparative HPLC, nisin fragments (1-12), (1-20), (22-34), and (22-31) could be isolated and will be used in a semi-synthesis approach to elucidate the role of each fragment in the mode of action of nisin as an antimicrobial peptide.
authors.affiliation: Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, PO Box 80082, 3508, TB, Utrecht, The Netherlands.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Slootweg
authors.forename: Rob M J
authors.initials: RM
authors.lastname: Liskamp
authors.forename: Dirk T S
authors.initials: DT
authors.lastname: Rijkers
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification*; D060326:Chemistry Techniques, Synthetic; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry; D003488:Cyanogen Bromide / Q000737:chemistry; D008969:Molecular Sequence Data; D009561:Nisin / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D009561:Nisin; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide
keywords: CNBr cleavage; LC-MS; antimicrobial peptides; enzymatic digestion; nisin; peptide purification; semi-synthesis