氨基酸分子改性的SiO_2杂化材料用于胰蛋白酶固定化

Immobilization of Trypsin on Amino Acid-modified SiO_2 Porous Materials

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,对水解前驱体3-氨基丙基三甲氧基硅烷进行了氨基酸分子改性.采用N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再与3-氨基丙基三甲氧基硅烷反应生成N-(3-三甲氧基硅基)丙基-N'-Fmoc-L-缬氨酰胺.然后去除Fmoc,得到N-(3-三甲氧基硅基)丙基-L-缬氨酰胺.通过IR,MS和1H NMR等分析手段对合成化合物的结构进行了表征.以正硅酸甲酯(TMOS)和N-(3-三甲氧基硅基)丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶法包埋胰蛋白酶,当N-(3-三甲氧基硅基)丙基-L-缬氨酰胺的摩尔分数为15%时,固定化胰蛋白酶活力的绝对值为199 U,游离酶的酶活力绝对值为103 U,而四甲氧基硅烷直接包埋的固定化酶活力的活性仅为38 U.由杂化硅源得到的固定化酶的活性是以四甲氧基硅烷为水解前驱体的固定化酶活性的5倍.杂化硅源固定化胰蛋白酶比游离酶的最高活力提高近2倍.结果表明,氨基酸分子经水解前驱体修饰后,水解产生的固定化载体具有良好的生物相容性.通过改性载体制备的固定化酶对甲醇变性剂的稳定性、酸碱的抵抗性及热稳定性均有明显提高.

To improve the biocompatibility and hydrophobicity of silicate,and to maintain the biological acti-vity of embedded biological molecules in silica glass,the hydrolysis precursor 3-aminopropyl trimethoxysilane was modified by L-valine.Specifically,the 3-methyl-2-（9-fluorenylmethoxycarbonylamino）butanoic acid reacted with thionyl chloride to produce 2-（9-fluorenylmethoxycarbonylamino）-3-methyl-butanoyl chloride.The obtained compound further reacted with 3-aminopropyl trimethoxysilane to obtain 2-（9-fluorenylmethoxycarbonylamino）-3-methyl-N-propyltrimethoxysilane butanamide.Finally,9-fluorenylmethoxycarbonyl（Fmoc） group was removed and the target compound 2-amino-3-methyl-N-propyltrimethoxysilane butanamide was synthesized.All obtained compounds were characterized by IR,MS,1H NMR.The immobilized trypsins were prepared on a mild sol-gel process.It shows the maximum biological activity while the molar fraction of 2-amino-3-methyl-N-propyltrimethoxysilane butanamide in hydrolysis precursor is 15%.The enzyme activity of immobilized trypsin by 2-amino-3-methyl-N-propyltrimethoxysilane butanamide is 2 times of that of free enzyme and is 5 times of that of enzyme immobilized by tetramethoxysilane.The results indicate that synthesized hybrid materials have a good biocompatibility.In addition,the materials show a good stability on denatured agent methanol,acidic and basic solution.The immobilized enzyme demonstrate an improved thermal stability either.