Huijbers Mieke M E, van Berkel Willem J H
Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.
Biotechnol J. 2015 Mar;10(3):395-403. doi: 10.1002/biot.201400229. Epub 2015 Jan 19.
Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to Δ(1) -pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.
脯氨酸脱氢酶（ProDH）催化脯氨酸在FAD依赖下氧化为Δ(1)-吡咯啉-5-羧酸，这是许多生物体中脯氨酸分解代谢的第一步。除了参与多种生理过程外，ProDH在实际应用中也备受关注，因为脯氨酸亚氨基酸可作为多种肽和抗生素的构建单元。ProDH是一种膜相关蛋白，仅获得了少量的重组可溶性形式的该酶。我们在此报告嗜热栖热菌（TtProDH）的脯氨酸脱氢酶在大肠杆菌中的异源表达。使用麦芽糖结合蛋白作为溶解性标签，可获得高产率的活性全酶。通过用胰蛋白酶切割纯化的融合蛋白可产生天然的TtProDH。尺寸排阻色谱显示融合和切割后的TtProDH形成寡聚体。热稳定性和共溶剂耐受性表明TtProDH的构象稳定性。这些特性与高产率一起使得TtProDH在工业应用中具有吸引力。
