Study On Natural Silicate Nanotubes As Supports For Immobilized Enzymes

The natural nanotubes HNT is an excellent aluminosilicate clay minerals with a hollow nanotubular structure and can be mined from the natural environment. In this paper, 100% yield of HNT can be obtained. In addition to the common advantages of inorganic supports for immobilized enzymes, HNT with both ends open possesses integrated morphology of hollow tubular structures and demonstrate unique characteristics, such as a remarkable pore structure that is more accessible for anchoring enzymes, a high specific surface area, adequate hydroxyl groups and good thermal stability. These characteristics give them a very high adsorption capability, making them promising candidates for the immobilization of enzymes. a-Amylase (EC 3.2.1.1) is a starch-hydrolyzed enzyme. It is an endo-enzyme that can cut a-l,4-glycosidic linkage of starch molecules and has been widely used in grain processing, fermentation and food industry, chemical engineering and so on. Urease (EC 3.5.1.5) can catalyze the hydrolysis of urea to form ammonia and carbon dioxide, and has great application foreground in biosensors, medical diagnosis as well as environmental monitoring. However, the application of them is often hampered by the short catalytic lifetime of enzymes and by the difficulty in recovery and recycling. The technology of enzyme immobilized can realize possibilities for storage, re-use and increased stability.In this paper, a-amylase and urease were immobilized on HNT with a physical adsorption method. Though study the concentration of enzymes, pH and reaction time, the conditions for a-amylase and urease immobilization were optimized. The properties of immobilized a-amylase and urease were also studied and compared with the free enzymes. The results further demonstrated that HNT can serve as an excellent support for enzyme immobilization.Firstly, the physical and chemical performance of the natural nanotubes HNT were investigated by means of Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and specific surface area measurements (SSA). The results showed that the HNT with adequate hydroxyl groups has a clearly hollow tubular structure, and an average length of 0.5～1.5μm, a diameter in the range of 30～50 nm, an average pore diameter of 10～30 nm, and a high specific surface area of 146.34 m /g; TG-DSC curves of HNT showed that they had excellent thermal stability and did not destroyed after calcination at 400℃for 2 h.Secondly, the optimized conditions for a-amylase and urease immobilization were obtained: 0.25 g HNT was added to 10 mL a-amylase solution (15 mg/mL, pH 6.07), then the mixture was shaken at room temperature for 5 h. The maximum adsorption amount of a-amylase by HNTs is 15.4 mg/g and the immobilization efficiency could achieve 37.38%; 0.25 g HNT was added to 10 mL urease solution (6 mg/mL, pH 7.0). The mixture was shaken at room temperature for 8 h. The maximum adsorption amount of urease by HNTs is 11.7 mg/g and the immobilization efficiency could achieve 33.13%.Finally, the properties of immobilized a-amylase and urease were tested and compared with the free enzymes. Upon immobilization, both the thermal stability and storage stability of the a-amylase and urease were improved. The immobilized a-amylase showed a 19% loss in activity at 80℃for 15 min and could retain about 90% of the original activity for 15 days (stored at 4℃). Furthermore, the immobilized a-amylase maintained over 56.2% of the original activity after 7 successive batch reactions.The immobilized urease showed a 12% loss in activity at 80℃for 15 min and could retain about 98.6% of the original activity for 15 days (stored at 4℃). Furthermore, after 10 successive batch reactions, the immobilized urease maintained over 65% of the original activity.