Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd, Taipei 10607, Taiwan.
Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd, Taipei 10607, Taiwan..
Int J Biol Macromol. 2019 Jul 15;133:980-986. doi: 10.1016/j.ijbiomac.2019.04.171. Epub 2019 Apr 25.
Hemoglobin A1c (HbA1c) is a hemoglobin molecule in which the N-terminal valine residue of the β subunit has been grafted with the glucose in blood. Its detection has important implications for the diagnosis of diabetes. Enzymatic colorimetric method using fructosyl peptide oxidase (FPO) is simple and rapid for HbA1c detection. A FPO mutant with enhanced activity was constructed and produced by E. coli; however, most of expressed mutant FPO was insoluble. Significantly enhanced expression solubility was achieved when cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of FPO mutant. Via the high affinity interaction between CBD and cellulose, the CBD fusion also facilitated the simultaneous purification and immobilization of FPO directly from E. coli cells lysate using bacterial cellulose (BC) nanofibrils as a matrix of very high specific area. A never-dried and water durable nanocellulose film with FPO activity could be easily obtained by collecting the FPO immobilized BC nanofibrils suspension on the surface of a microfiltration membrane. The activity of the ready-use FPO nanocellulose film was stable at least 7 days at room temperature for the detection of HbA1c level of 5.3-11% in blood samples.
血红蛋白 A1c（HbA1c）是血红蛋白分子中β亚基的 N 端缬氨酸残基与血液中的葡萄糖结合而成的。它的检测对糖尿病的诊断具有重要意义。使用果糖肽氧化酶（FPO）的酶比色法检测 HbA1c 简单快速。通过大肠杆菌构建并生产了一种活性增强的 FPO 突变体；然而，大多数表达的突变 FPO 是不溶的。当将纤维素结合结构域（CBD）从热纤维梭菌融合到 FPO 突变体的 N 端时，可显著提高表达的可溶性。通过 CBD 与纤维素之间的高亲和力相互作用，CBD 融合还促进了 FPO 从大肠杆菌细胞裂解物中直接通过细菌纤维素（BC）纳米纤维进行同时纯化和固定，BC 纳米纤维作为高比表面积的基质。通过将固定化 FPO 的 BC 纳米纤维悬浮液收集在微滤膜表面上，很容易获得具有 FPO 活性的、永不干燥且耐水的纳米纤维素薄膜。这种即用型 FPO 纳米纤维素薄膜的活性在室温下至少稳定 7 天，可用于检测血液样本中 5.3-11%的 HbA1c 水平。
