用于α-胰凝乳蛋白酶固定化的氨基超顺磁纳米凝胶的光化学合成与表征

Photochemical Synthesis and Characterization of Amine-functionalized Superparamagnetic Nanogel for α-Chymotrypsin Immobilization

通过霍夫曼降解光化学原位聚合制备的聚丙烯酰胺包覆的Fe3O4纳米粒子得到了氨基化磁性纳米凝胶,用缩合剂1-乙基-3-(3-二甲胺)碳二亚胺成功地将α-胰凝乳蛋白酶固定到氨基化磁性纳米凝胶上,并采用光子相关光谱、傅里叶变换红外光谱、透射电子显微镜、X射线粉末衍射和热重-示差扫描量热联用等多种手段对其进行了表征.固定化了的α-胰凝乳蛋白酶平均粒径约为31 nm;热重法测得每克凝胶上的载酶量为69 mg,BCA法测得每克凝胶上的载酶量为61 mg;酶的固定化和氙灯辐照并未改变Fe3O4的晶形结构;固定化酶比活力为0.93 U/(mg.m in),为自由酶活力的59.3%;磁含量高达88%,具有优异的磁响应性能,可应用于诸多生物医药领域的快速检测、分离及酶的再生利用.

In this paper, amine-functionalized superparamagnetic nanogel was obtained by Hoffman degradation of the PAM-coated Fe3O4 nanoparticles prepared by photochemical in situ polymerization, α-Chymotrypsin was successfully bound to the magnetic nanogel with amino groups in the presence of 1-ethyl-3-（3-dimethylaminepropyl） carbodiimide （EDC）. The immobilized enzyme was characterized by using photo correlation spectroscopy（PCS）, transmission electron microscopy（TEM）, X-ray diffraction （XRD） analysis, thermogravimetric-differential scanning calorimetry （ TG-DSC ） and Fourier transform infrared spectroscopy. Mean particle size of the immobilized enzyme was measured to be 31 nm by PCS; the binding capacity was 69 mg enzyme/g nanogel determined by TG analysis, 61 mg enzyme /g nanogel determined by standard BCA. The crystalline structure of Fe3O4 was affected by xenon lamp irradiation and enzyme immobilization. Magnetic content of the immobilized enzyme was as high as 88%. Specific activity of the immobilized α-chymotrypsin was 0. 93 U/min ·mg）, 59.3% of free form. It can be expected that the immobilized α-chymotrypsin has a potential in the fast detection and separation, as well as the reuse of immobilized enzymes.