An efficient method for 15N-labeling of chitin in fungi

To permit facile (15)N solid-state NMR (ssNMR) analysis of the degree of acetylation (DA) of chitinous materials in fungi a method for the introduction of a (15)N isotopic label has been developed. Using Penicillium chrysogenum as a model system, a series of (15)N-based media were surveyed for their abilities to support mycelial growth, and a rich medium supplemented with ((15)NH(4))(2)SO(4) supported good growth. Uptake of label into chitin extracted from mycelia grown in the rich ((15)NH(4))(2)SO(4)-based media was monitored by mass spectrometry, with approximately 1 g/L of ((15)NH(4))(2)SO(4) leading to approximately 65% incorporation. The labeled chitin was studied by ssNMR to determine its DA, and the (15)N label permitted measurement of DA to within 0.5% with acquisition times of on the order of half an hour. Similar studies validated the method for DA measurements on chitin from cultures of Aspergillus niger and Mucor rouxii.