Abstract
Pseudoalteromone A (PA) is a cytotoxic and anti-inflammatory ubiquinone discovered recently from a marine bacterium Pseudoalteromonas sp. CGH2XX. In order to meet its sample supply for further in vivo pharmacological investigation, an efficient method was developed for the preparation of PA by combination of response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC) from marine bacterium P. rubra QD1-2. First, optimization of culture conditions was studied by the RSM to enhance PA production. The results indicated that the optimal cultivation condition was peptone (2.21 g/l), yeast extract (3.125 g/l), glucose (0.125 g/l), KBr (0.02 g/l), inoculum size (6.5 %), medium volume (595 ml), initial pH value (7.0), temperature (28 °C). Under the optimized fermentation condition, PA production was 1.04 mg/l with 14.8-fold increase comparing to 0.07 mg/l under original standard fermentation condition. The PA production was further investigated using a 14-l jar fermenter. Compared to the flask culture, P. rubra QD1-2 offered 45 % increase of PA production at 1.51 mg/l. Then, a rapid and efficient method for the separation and purification of PA from crude culture extract was developed using HSCCC. The two-phase solvent system used for HSCCC separation was composed of n-hexane–ethyl acetate–methanol–water (5:5:9:5, v/v/v/v). The isolation was accomplished within 100 min, and the purity of PA was over 95 %. The recovery of the process was 93 %.
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Acknowledgments
This work was financially supported by National Natural Science Foundation of China (41306134), 863 Program of China (2013AA092902), Zhejiang Provincial Natural Science Foundation of China (LQ13B020004), Ph.D. Programs Foundation of Ministry of Education of China (20133305120007), Natural Science Foundation of Ningbo (2013A610278), Zhejiang Marine Biotechnology Innovation Team (2012R10029-2), Ningbo Marine Algae Biotechnology Team (2011B81007), Scientific Research Foundation for Returned Scholars of ZJHRSS and K.C. Wong Magna Fund in Ningbo University.
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Ding, L., He, S. & Yan, X. Efficient preparation of pseudoalteromone A from marine Pseudoalteromonas rubra QD1-2 by combination of response surface methodology and high-speed counter-current chromatography: a comparison with high-performance liquid chromatography. Appl Microbiol Biotechnol 98, 4369–4377 (2014). https://doi.org/10.1007/s00253-014-5530-0
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DOI: https://doi.org/10.1007/s00253-014-5530-0