通过在双水相体系中进行分配来纯化重组苯丙氨酸脱氢酶。
Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems.
作者信息
Mohamadi Hamid Shahbaz, Omidinia Eskandar
机构信息
Young Researcher Club, Department of Biochemistry, Science & Research Campus, Islamic Azad University, Tehran, Iran.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):273-8. doi: 10.1016/j.jchromb.2007.04.049. Epub 2007 May 16.
This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.
本研究介绍了在由聚乙二醇6000（PEG - 6000）和硫酸铵组成的双水相体系（ATPS）中重组巴氏芽孢杆菌苯丙氨酸脱氢酶（PheDH）的分配和纯化。开发了一种双水相体系的单步操作方法，用于从大肠杆菌BL21（DE3）中提取和纯化重组PheDH。研究了体系参数，包括PEG分子量和浓度、pH值、硫酸铵浓度以及氯化钠添加量对酶分配的影响。用于PheDH分配和纯化的最佳体系为pH 8.0时，8.5%（w/w）的PEG - 6000、17.5%（w/w）的硫酸铵和13%（w/w）的氯化钠。分配系数、回收率、产率、纯化倍数和比活性值分别为92.57、141%、95.85%、474.3和10424.97 U/mg。此外，氧化脱氨反应中L - 苯丙氨酸和NAD⁺的米氏常数（Kₘ）值分别为0.020和0.13 mM。我们的数据表明，这种双水相体系可能是一种用于大规模纯化重组PheDH的经济且有吸引力的技术。
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