Çakıroğlu Bekir, Çiğil Aslı Beyler, Ogan Ayşe, Kahraman M Vezir, Demir Serap
Biomedical, Magnetic, and Semiconductor Materials Research Center (BIMAS-RC) Sakarya University Sakarya Turkey.
Department of Chemistry Faculty of Arts and Sciences Marmara University Istanbul Turkey.
Eng Life Sci. 2018 Jan 29;18(4):254-262. doi: 10.1002/elsc.201700130. eCollection 2018 Apr.
In this study, polyacrylic acid-based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from was covalently immobilized onto polyacrylic acid-based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid-based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn, Ni, Cu, Zn, Mg, Ca ions on the stability of the immobilized AChE was also investigated. According to the Michaelis-Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis-Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE-based biosensors.
在本研究中，通过静电纺丝法制备了聚丙烯酸基纳米纤维（NF）膜。通过高效的酶固定化方法，将源自[具体来源未给出]的乙酰胆碱酯酶（AChE）共价固定在聚丙烯酸基NF膜上，发现聚合物膜的固定化容量为0.4 mg/g。通过热活化表面重构以及静电纺丝后用羰基二咪唑进行活化，合成了新型NF膜。通过扫描电子显微镜、傅里叶变换红外光谱和热重分析对聚丙烯酸基膜的形态进行了研究。研究了温度和pH对酶活性的影响，游离酶和固定化酶的最大活性分别在30和35°C以及pH 7.4和8.0时观察到。还研究了1 mM的Mn、Ni、Cu、Zn、Mg、Ca离子对固定化AChE稳定性的影响。根据米氏方程曲线，固定化后AChE对碘化硫代乙酰胆碱的亲和力较低，固定化AChE和游离AChE的米氏常数分别为0.5008和0.4733 mM。固定化AChE表现出令人满意的可重复使用性，即使在连续进行10次活性测定后，AChE仍保持其初始活性的约87%。游离酶在60天内完全失去活性，而固定化酶在相同储存时间下保留了约70%的初始活性。固定化AChE良好的可重复使用性使得该载体可用于开发基于AChE的生物传感器。
