天然硅酸盐纳米管作为酶固定化载体的研究
摘要
天然纳米管HNT是一种天然优良的粘土质硅酸盐矿物,在天然条件下由硅酸盐片层卷曲而成的多壁微管状结构。本文中的HNT纳米管产率极高,接近100%,作为酶固定化的载体材料,它具有传统无机材料的所有优势;另外,HNT为形态完整的中空管状结构,纳米管两端开口,尺寸分布均匀稳定,具有良好的热稳定性和与酶分子相适宜的孔直径范围,而且HNT纳米管高的比表面积具有许多优越的吸附性能,富含的羟基官能团也有利于提高酶的固载率。因此HNT是一种极具潜力和竞争力的固定化酶载体材料。a-淀粉酶(EC 3.2.1.1)是一种内切葡萄糖苷酶,是目前最重要的工业酶制剂之一,广泛应用于粮食加工、食品工业、发酵工业、纺织品工业和医药工业等领域。脲酶(EC 3.5.1.5)能将尿素水解为氨和二氧化碳,它在生物传感器、医疗诊断、环境监测等诸多方面有着广阔的应用前景。但是游离酶对环境十分敏感,活性不易保持,且回收困难,使用成本高。酶固定化技术是实现酶重复连续使用和稳定性改善的有效手段。
本文以新型天然纳米材料HNT作为酶固定化的载体材料,采用物理吸附法,制备固定化a-淀粉酶和固定化脲酶,通过测定酶活和固载率,从给酶量、pH值、固定化时间三个因素优化了固定化条件,研究了固定化酶的酶学性能,并与游离酶进行了比较。结果表明HNT具有稳定的物理化学性能,是一种良好的酶固定化载体材料。
本文首先研究了天然纳米管HNT的物理化学性能。利用Fourier红外光谱、TEM、SEM和比表面及空隙率测定仪等对其进行表征,结果表明HNT富含羟基官能团,为中空管状结构,长度在0.5～1.5μm之间,管外径约30～50nm,管内径约10～30 nm,比表面积可达146.34 m~2/g;利用Netzsch热分析仪进行DSC-TG分析,研究表明HNT具有良好的热稳定性能,在400℃×2h煅烧数次循环未发现纳米管被破坏。
其次,对以HNT为载体固定化a-淀粉酶和脲酶的最佳条件进行了研究。结果表明:0.25 g HNT细粉与10 mL浓度为15 mg/mL的a-淀粉酶酶液(由pH=6.07的缓冲溶液配制)固定结合,室温下振荡5 h后,酶的固定化效率最好,最大吸附量为15.4 mg/gHNT,固载率平均达到37.38%;用0.25 g HNT细粉与10 mL浓度为6 mg/mL的脲酶酶液(由pH=7.0的缓冲溶液配制)固定结合,室温下振荡8 h后,酶的固定化效率最好,最大吸附量为11.7 mg/gHNT,固载率平均达到33.13%。
最后,研究了固定化酶的性质,并与游离酶进行了比较,结果表明:固定化酶的热稳定性和贮藏稳定性明显优于游离酶;固定化a-淀粉酶在80℃下保温15min后酶活仅下降19%左右;在4℃下保存15 d,仍保持90%的催化活性,并且连续7批次操作后其相对活力保持在56.2%左右,显示出良好的稳定性。
固定化脲酶在80℃下保温15 min后酶活仅下降12%左右;4℃下保存15 d后,酶活力基本没有下降,并且连续10批次操作后其相对活力保持在65%左右,同样显示出良好的稳定性。
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.
本文以新型天然纳米材料HNT作为酶固定化的载体材料,采用物理吸附法,制备固定化a-淀粉酶和固定化脲酶,通过测定酶活和固载率,从给酶量、pH值、固定化时间三个因素优化了固定化条件,研究了固定化酶的酶学性能,并与游离酶进行了比较。结果表明HNT具有稳定的物理化学性能,是一种良好的酶固定化载体材料。
本文首先研究了天然纳米管HNT的物理化学性能。利用Fourier红外光谱、TEM、SEM和比表面及空隙率测定仪等对其进行表征,结果表明HNT富含羟基官能团,为中空管状结构,长度在0.5～1.5μm之间,管外径约30～50nm,管内径约10～30 nm,比表面积可达146.34 m~2/g;利用Netzsch热分析仪进行DSC-TG分析,研究表明HNT具有良好的热稳定性能,在400℃×2h煅烧数次循环未发现纳米管被破坏。
其次,对以HNT为载体固定化a-淀粉酶和脲酶的最佳条件进行了研究。结果表明:0.25 g HNT细粉与10 mL浓度为15 mg/mL的a-淀粉酶酶液(由pH=6.07的缓冲溶液配制)固定结合,室温下振荡5 h后,酶的固定化效率最好,最大吸附量为15.4 mg/gHNT,固载率平均达到37.38%;用0.25 g HNT细粉与10 mL浓度为6 mg/mL的脲酶酶液(由pH=7.0的缓冲溶液配制)固定结合,室温下振荡8 h后,酶的固定化效率最好,最大吸附量为11.7 mg/gHNT,固载率平均达到33.13%。
最后,研究了固定化酶的性质,并与游离酶进行了比较,结果表明:固定化酶的热稳定性和贮藏稳定性明显优于游离酶;固定化a-淀粉酶在80℃下保温15min后酶活仅下降19%左右;在4℃下保存15 d,仍保持90%的催化活性,并且连续7批次操作后其相对活力保持在56.2%左右,显示出良好的稳定性。
固定化脲酶在80℃下保温15 min后酶活仅下降12%左右;4℃下保存15 d后,酶活力基本没有下降,并且连续10批次操作后其相对活力保持在65%左右,同样显示出良好的稳定性。
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.
引文
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