Schwabe Ringo, Anke Marlene Kirstin, Szymańska Katarzyna, Wiche Oliver, Tischler Dirk
Institute of Biological Sciences, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Straße 29, 09599, Freiberg, Germany.
Department of Chemical Engineering and Process Design, Silesian University of Technology, Ks. M. Strzody 7, 44-100, Gliwice, Poland.
Res Microbiol. 2018 Dec;169(10):598-607. doi: 10.1016/j.resmic.2018.08.002. Epub 2018 Aug 20.
The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe > Ga > V > Al) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga- and Nd-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores.
放线菌戈登氏红染菌CWB2（DSM 46758）在铁限制条件下产生异羟肟酸型铁载体（188毫克/升）。分析型反相高效液相色谱法可测定培养液中三价铁螯合化合物的单一峰，这通过Fe-CAS测定得到了证实。洗脱曲线及其吸收光谱与用作参考化合物的市售（去）铁胺B相似。这证实了先前的假设，并首次表明戈登氏菌属产生类铁胺铁载体。对反相高效液相色谱方法进行了优化，以分离无金属和负载金属的草氨酸。这使得能够测定溶液中铁载体的浓度以及金属亲和力。通过电感耦合等离子体质谱法确认了草氨酸的金属负载情况。结果表明，铁胺优先结合三价金属离子（Fe＞Ga＞V＞Al）而非二价金属离子。此外，我们旨在展示新建立的反相高效液相色谱方法的适用性，并通过固定在介孔二氧化硅泡沫载体上来提高铁胺作为金属螯合剂的可重复使用性。从菌株CWB2和市售铁胺B获得的铁载体成功地以高产率（高达95%）与载体相连，这通过高效液相色谱法得到了验证。金属结合研究表明，金属可以与未固定的和共价连接的铁胺结合，也可以与载体材料本身结合。发现后者是非特异性的，因此载体材料的影响仍是未来研究的一个领域。通过针对各种元素（Nd、Gd、La、Er、Al、Ga、V、Au、Fe、As）的反向CAS测定，有可能证明负载铁胺的介孔二氧化硅载体对Ga和Nd的结合得到了改善。强大的反相高效液相色谱方法与各种CAS测定的结合为筛选产铁载体菌株以及控制铁载体的纯化和固定提供了新途径。
