Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase |
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| Authors: | Eskandar Omidinia Hamid Shahbaz Mohamadi Rassoul Dinarvand Heshmat-Allah Taherkhani |
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| Institution: | (1) Department of Biochemistry, Pasteur Institute of Iran, 13164 Tehran, Iran;(2) Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran;(3) Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran; |
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| Abstract: | This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process
for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K2HPO4 ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of
chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K2HPO4 and 16% (w/w) KCl with pH of 8.0, volume ratio (V
R
) of 0.25, temperature of 25 °C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process.
A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process
time (4 vs. 48 h) and the lower total cost (4 vs. 20 €) were additionally features that confirmed the suitability of proposed
technique. |
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