CO差光谱法分析重组大肠杆菌中的透明颤菌血红蛋白

Carbon monoxide difference spectrum analysis of Vitreoscilla hemoglobin in recombinant E.coli

为检测产聚 β-羟基丁酸酯 (PHB)的重组大肠杆菌VG1(p TU14)中表达的透明颤菌血红蛋白 (VHb) ,建立了改进的 CO差光谱法。结果表明 ,PHB对 VHb的检测有较严重的干扰。 VG1(p TU14)的全细胞悬浊液经过 4次冻融、低速离心和过量的 Na2 S2 O4还原后 ,在试样端通入 CO进行3m in络合反应并在黑暗条件下静置 5 min,最后进行 VHb的 CO差光谱分析。当菌浓小于 2 5 m g/ m L缓冲液时 ,其定量关系符合 L am ber- Beer定律 ,可以准确地定量分析

An improved method using the carbon monoxide (CO) difference spectrum was used to analyze Vitreoscilla hemoglobin (VHb) in recombinant Escherichia coli, VG1 (pTU14), producing poly-β-hydroxybutyrate (PHB). The PHB granules caused heavy interference with the VHb spectra. The interference was eliminated using four freeze-thaw cycles, centrifugation at low speed, and excess sodium dithionite treatment. Then CO gas was bubbled into the whole cell suspension of VG1 (pTU14) for 3 min to coordinate with VHb and the suspension was placed in the dark for 5 min for better spectra. The Lambert-Beer law can be used to quantitatively analyze VHb when the cell concentration is less than 25 mg/mL.