Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Rd. Sec 4, Taipei 106, Taiwan.
Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Rd. Sec 4, Taipei 106, Taiwan.
Int J Biol Macromol. 2018 Dec;120(Pt A):325-331. doi: 10.1016/j.ijbiomac.2018.08.096. Epub 2018 Aug 21.
Recombinant fructosyl peptide oxidase (FPO) from Phaeosphaeria nodorum SN15 was functionally expressed by Escherichia coli cells and one-step purified from crude cells extract using immobilized metal affinity chromatography (IMAC) to achieve a specific activity of 26 U/mg. A ready-use colorimetric detection of HbA1c level in blood sample was developed based on FPO immobilized membrane. Facile bio-inspired polydopamine coating on the surface of a microporous membrane was employed for effective FPO immobilization. Glutaraldehyde activation of the polydopamine coating significantly enhanced FPO immobilization yield that at least 5-fold higher activity could be achieved. The stability of FPO membrane was also enhanced by glutaraldehyde activation that 85% activity could be maintained after 7 repeated uses. Highly correlated optical densities at 727 nm (OD) against fructosylvaline (FV) in the range of 0.02 mM to 0.7 mM (R = 0.988) could be achieved using FPO membrane. At least 80% of the initial activity was maintained for FPO membrane stored at 4 °C for 7 days. Rather low OD but good correlation still could be obtained by using FPO membrane for the detection HbA1c levels (6-14%) in blood samples.
从禾谷镰刀菌 SN15 中重组得到的果糖基肽氧化酶（FPO）在大肠杆菌细胞中实现了功能表达，并通过固定金属亲和层析（IMAC）从粗细胞提取物中一步纯化，达到了 26 U/mg 的比活。基于固定化 FPO 的膜，开发了一种用于血液样本中 HbA1c 水平的即用型比色检测方法。在微孔膜表面采用简便的仿生聚多巴胺涂层，实现了有效的 FPO 固定化。聚多巴胺涂层的戊二醛活化显著提高了 FPO 的固定化产率，可实现至少 5 倍的高活性。戊二醛活化还增强了 FPO 膜的稳定性，经过 7 次重复使用后，仍能保持 85%的活性。使用 FPO 膜，在 0.02-0.7 mM 的果糖基缬氨酸（FV）范围内，可实现 727nm 处的高相关光学密度（OD）（R = 0.988）。FPO 膜在 4°C 下储存 7 天，仍能保持至少 80%的初始活性。即使使用 FPO 膜检测血液样本中的 HbA1c 水平（6-14%），OD 值虽然较低，但相关性仍然良好。
