Zinc oxide nanoparticles-impregnated chitosan surfaces for covalent immobilization of trypsin: Stability & kinetic studies.
Zinc oxide nanoparticles-impregnated chitosan surfaces for covalent immobilization of trypsin: Stability & kinetic studies.
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DOI：
10.1016/j.ijbiomac.2022.03.014
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发表时间：
2022-03
影响因子：
8.2
通讯作者：
Shalu Aggarwal;S. Ikram
中科院分区：
文献类型：
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作者：
Shalu Aggarwal;S. Ikram
胰蛋白酶（Try， EC）3.4.21.4)通过席夫碱键共价固定在戊二醛（GA）活化的ZnO/壳聚糖纳米复合材料表面。采用紫外可见光谱、傅里叶变换红外光谱（FTIR）、x射线衍射分析（XRD）、透射电子显微镜（TEM）、扫描电子显微镜（SEM）和能量色散x射线微分析（EDAX）等技术对制备的ZnO纳米颗粒和壳聚糖包覆ZnO纳米复合材料的尺寸、结构、表面形貌和元素组成百分比进行了研究。研究了最佳固定化条件（孵育时间16 h，酶浓度1.8 mg/ml， pH值7.8）以获得固定化胰蛋白酶的最大表达活性。固定化和溶解胰蛋白酶分别在pH为8.5、60°C和pH为7.8、45°C时表现出最佳的催化活性。测定了固定化（27.12 μM, 8.82 μM/min）和游离胰蛋白酶（25.76 μM, 4.16 μM/min）的动力学参数Km， Vmax，表明固定化后胰蛋白酶的效率有所提高。固定化胰蛋白酶在50°C孵育2小时内保持67%的初始活性，并保持近50%的催化活性，直到第9次重复循环利用。此外，固定化胰蛋白酶在4°C下保存90天后仍保持50%的酶活性。因此，目前的研究结果表明，ZnO/壳聚糖- ga -胰蛋白酶将是一种有前景的大规模生物技术应用的生物催化剂。
Trypsin (Try, EC. 3.4.21.4) was effectively immobilized on the surface of glutaraldehyde(GA)-activated ZnO/Chitosan nanocomposite through covalent attachment via Schiff-base linkages. Size, structure, surface morphology, & percentage elemental composition of the prepared ZnO nanoparticles and chitosan-coated ZnO nanocomposite were studied by UV–Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-Ray diffraction analysis (XRD), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), and Energy-Dispersive X-Ray Microanalysis (EDAX) techniques. Optimal immobilization conditions (incubation time (16 h), enzyme concentration (1.8 mg/ml), and pH (7.8)) were investigated to obtain the maximum expressed activity of the immobilized trypsin. Immobilized & solubilized trypsin exhibited the optimum catalytic activity at pH 8.5, 60 °C, and pH 7.8, 45 °C respectively. Kinetic parameters (Km, Vmax) of immobilized (27.12 μM, 8.82 μM/min) & free trypsin (25.76 μM, 4.16 μM/min) were determined, indicating that efficiency of trypsin improves after immobilization. Immobilized trypsin preserved 67% of initial activity at 50 °C during 2 h of incubation & sustained nearly 50% of catalytic activity until the 9th repeated cycle of utilization. Moreover, immobilized trypsin retained 50% of enzymatic activity after 90 days of storage at 4 °C. Hence, the current findings suggest that ZnO/Chitosan-GA-Trypsin would be a promising biocatalyst for large-scale biotechnological applications.