Ahmad Razi, Sardar Meryam
Indian J Biochem Biophys. 2014 Aug;51(4):314-20.
Immobilization of cellulase from Aspergillus niger on TiO2 nanoparticles was studied by two different approaches--physical adsorption and covalent coupling. A. niger was selected, as it is generally non-pathogenic, is found in nature in the broad range of habitats and produces cellulase extracellulary. For covalent method, TiO2 nanoparticles were modified with aminopropyltriethoxysilane (APTS). The adsorbed and covalently immobilized enzymes showed 76% and 93% activity, respectively, as compared to the free enzyme. The catalytic efficiency V(max)/K(m) increased from 0.4 to 4.0 after covalent attachment, whereas in adsorption method, it increased slightly from 0.4 to 1.2. The covalently-immobilized and adsorbed cellulase lost only 25% and 50% of their activity, respectively after 60 min of incubation at 75 degrees C. The reusability and operational stability data also showed that covalent coupling increased the stability of the enzyme. The presence of enzyme on TiO2 nanoparticles was confirmed by Fourier-transform infrared spectroscopy. The high-resolution transmission electron microscopy (HR-TEM) and atomic force microscopy (AFM) studies indicated aggregation of enzyme when adsorbed on TiO2 surface and a monolayer of enzyme in covalent attachment. In conclusion, covalently attached cellulase retained good activity and thermal stability, as compared to physically adsorbed enzyme. The lower amount of enzyme activity and thermal stability in case of physically adsorbed immobilized enzyme was due to aggregation of the enzyme after adsorption on TiO2 nanoparticles, as revealed by HR-TEM and AFM. Thus, TiO2 nanoparticles could be suitable candidates for immobilization of cellulase for industrial applications like paper, textile, detergent and food industries.
采用两种不同方法——物理吸附和共价偶联，研究了黑曲霉纤维素酶在二氧化钛纳米颗粒上的固定化。选择黑曲霉是因为它通常无致病性，在自然界广泛的栖息地中都能找到，且能在细胞外产生纤维素酶。对于共价法，用氨丙基三乙氧基硅烷（APTS）对二氧化钛纳米颗粒进行改性。与游离酶相比，吸附和共价固定化的酶活性分别为76%和93%。共价连接后催化效率V(max)/K(m)从0.4提高到4.0，而在吸附法中，仅从0.4略有提高到1.2。在75℃孵育60分钟后，共价固定化和吸附的纤维素酶分别仅损失其活性的25%和50%。可重复使用性和操作稳定性数据也表明共价偶联提高了酶的稳定性。通过傅里叶变换红外光谱证实了二氧化钛纳米颗粒上存在酶。高分辨率透射电子显微镜（HR-TEM）和原子力显微镜（AFM）研究表明，吸附在二氧化钛表面时酶会聚集，而共价连接时酶形成单层。总之，与物理吸附的酶相比，共价连接的纤维素酶保留了良好的活性和热稳定性。如HR-TEM和AFM所示，物理吸附固定化酶的酶活性和热稳定性较低是由于酶吸附在二氧化钛纳米颗粒后发生聚集。因此，二氧化钛纳米颗粒可能是用于造纸、纺织、洗涤剂和食品工业等工业应用中固定化纤维素酶的合适候选材料。
