The Studies On Siderophore Produced By The Marine Yeast Aureobasidium Pullulans HN6.2

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore.Among them,a total of 14 yeast strains were found to produce siderophore, and the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. The optimal medium components and growth conditions for siderophore production by strain HN6.2 were studied using"one-variable-at-a-time"method. The results showed that the optimal medium components for the highest siderophore yield by strain HN6.2 were sucrose 3.0% (w/v), NH₄NO₃ 0.3% (w/v),K₂HPO₄ 0.3% (w/v),citric acid 0.1%(w/v),MgSO₄ 0.008% (w/v),ZnSO₄ 0.0002% (w/v),10 mM L-ornithine and the optimal growth conditions were shaking speed 200 rpm, initial pH 6.2, temperature 28℃. Under these conditions, the siderophore in the liquid medium reached the highest yields 1.1mg/mL after 120h of fermentation. In this study, we found that addition of L-ornithine in the production medium could enhance the siderophore production by HN6.2, whereas the supplement of excess Fe3+ in the production medium could decrease the siderophore production. In the molecular level, the expression of the gene of hydroxylase was also repressed in the presence of Fe3+, The expression of the gene was stimulated in the presence of L-ornithine.We detected anti-microbial activity of the crude siderophore produced by strain HN6.2 by the Oxford-cups and colony-counting.The results showed that the crude siderophore had clear antibacterial activity against the two marine animal pathogens, V. anguillarum and V. parahaemolyticus and the common bacterium B. subtilis . However, the crude siderophore had no antimicrobial activity against the most common bacteria, E. coli, S. aureus, the M. bicuspidate WCY, two fungi A. niger and Penicillius sp. The results also showed that the heated siderophore still had the same antibacterial activity against B. sublitis, V. anguillarum and V. parahaemolyticus.In this study, the crude siderophore was extracted from the culture supernatants by using organic solvents, then,the crude siderophore was purified by reverse phase high pressuer liquid chromatography(RP-HPLC),and the aim peak was collected. The aim peak was analyzed by using the mass spectrum, and according to the molecular ion peak of the component, comparing with the foregone structure of the siderophore, the structure of the siderophore produced by strain HN6.2 was identified and was found to be fusigen which belongs to one kind of the hydroxamates.