Optimization of chitosan degrading enzyme production in the fungus Alternaria alternata by modifying the growing culture conditions

J Beneteau, S Chachra, NE El Gueddari, BM Moerschbacher
Department of Plant Biochemistry and Biotechnology, Westphalian Wilhems-University Münster, Schlossplatz 8, 48143 Münster, Germany
A chitosan degrading enzyme has been identified in the fungus Alternaria alternata. This chitinosanase is able to cleave chitosan and presents a high and unique specificity for the glycosidic linkage between a GlcNAc and a GlcN unit. In order to identify the protein sequence, a high amount of pure protein is required to perform de novo sequencing by mass spectrometry experiments. This protein is secreted in the media, and because of the low amount of protein produced, the presence of the protein can be followed only via the measurement of its activity. To increase the production of the protein, the fungus was grown under different culture conditions. 
A. alternata was cultivated in liquid media in presence of carbon sources such as sucrose and glucose, and in presence of different concentrations of chitosan. These experiments have shown that the protein expression is induced in the presence of chitosan, in a dose-dependent way, until a concentration at which the chitosan seems to be toxic for the fungus. Furthermore the expression of the protein is increased in presence of sucrose or glucose and chitosan is probably not used as a carbon source by the fungus. These results should allow us to obtain a sufficient amount of pure protein to identify its sequence.