Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran.
Mol Biotechnol. 2023 Nov;65(11):1809-1823. doi: 10.1007/s12033-023-00687-1. Epub 2023 Feb 16.
The immobilized enzymes' properties can be affected by cross-linkers on the surface of supports. To study how cross-linkers alter enzymes function, chitosan-coated magnetic nanoparticles (CMNPs) with immobilized papain were prepared using glutaraldehyde and or genipin, and then, the properties of the nanoparticles and the immobilized enzymes were assessed. The Scanning Electron Microscope (SEM), Fourier Transform Infrared (FTIR), and X-Ray Diffraction (XRD) results showed that the CMNPs were prepared and papain molecules were immobilized on CMNPs by glutaraldehyde (CMNP-Glu-Papain) or by genipin (CMNP-Gen-Papain). Also, the results associated with enzymes activity indicated that the immobilization by glutaraldehyde and genipin increased the pH optimum of papain from 7 to 7.5 and 9, respectively. The kinetic results indicated that the immobilization by genipin slightly affects the enzyme affinity to the substrate. The stability results showed that CMNP-Gen-Papain has more thermal stability than CMNP-Glu-Papain and papain immobilization on CMNPs by genipin leads to stabilization of the enzyme in the presence of polar solvents, probably due to the more hydroxyl groups on CMNPs activated by genipin. In conclusion, this study suggests that there is a relationship between the types of cross-linker on the surface of supports, and the mechanism of action, kinetic parameters, and the stability of immobilized papain.
固定化酶的性质可能会受到载体表面交联剂的影响。为了研究交联剂如何改变酶的功能，使用戊二醛和京尼平制备了壳聚糖包覆的磁性纳米粒子（CMNPs）固定化木瓜蛋白酶，并对纳米粒子和固定化酶的性质进行了评估。扫描电子显微镜（SEM）、傅里叶变换红外（FTIR）和 X 射线衍射（XRD）结果表明，CMNPs 被制备，并且木瓜蛋白酶分子通过戊二醛（CMNP-Glu-Papain）或京尼平（CMNP-Gen-Papain）固定在 CMNPs 上。此外，与酶活性相关的结果表明，戊二醛和京尼平固定化分别将木瓜蛋白酶的 pH 最适值从 7 提高到 7.5 和 9。动力学结果表明，京尼平固定化略微影响酶对底物的亲和力。稳定性结果表明，CMNP-Gen-Papain 比 CMNP-Glu-Papain 具有更高的热稳定性，并且京尼平固定在 CMNPs 上的木瓜蛋白酶在极性溶剂存在下稳定，这可能是由于京尼平激活的 CMNPs 上有更多的羟基。总之，本研究表明载体表面交联剂的类型与固定化木瓜蛋白酶的作用机制、动力学参数和稳定性之间存在关系。
