Purification of an intracellular metallopeptidase of M sub(r) 45000 in Fusarium moniliforme

• Published in: Mycological research 1997-06, Vol.101 (6), p.678-682
• Main Authors: Rodier, M H, El Moudni, B, Kauffman-Lacroix, C, Jacquemin, J L
• Format: Article
• Language: English
• Subjects: Fusarium moniliforme
• ISSN: 0953-7562

Cytoplasmic extracts of Fusarium moniliforme contained a peptidase activity, able to cleave preferentially L-Leu-7-amino-4-methylcoumarin fluorogenic substrate. No activity towards this substrate was detected in various culture media of F. moniliforme supplemented or not with different substrates (bovine serum albumin, bovine haemoglobin, collagen or gelatin), proving that the peptidase was not secreted in these conditions. The cytoplasmic enzyme was purified by high performance liquid chromatography using a combination of an anionic exchange and gel filtration columns. The purified activity gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, estimated at M sub(r) 45000 under reducing conditions. The aminopeptidase showed an optimum activity at pH 7,2, an isoelectric point at 4,1, the Michaelis constant was at 50 mu M and the V sub(max) at 12 mM AMC released min super(-1) mg super(-1) of protein for L-Leu-AMC. Since this natural peptidase is sensitive to the protease inhibitors 1,10-phenantroline and ethylenediaminetetraacetic acid, it is considered as a metallopeptidase.