Fe_3O_4磁性纳米粒子固定化胰蛋白酶

Immobilizing Trypsin Onto Micro Magnetic Particles for Protein Digestion

以Fe3O4@mSiO2@nSiO2介孔材料为载体,γ-氨丙基三乙氧基硅烷接枝,戊二醛交联,研究胰蛋白酶的固定化及其在蛋白酶解中的应用。对蛋白酶固定化条件的优化研究表明,φ(戊二醛)=1.875%、给酶量为0.3 mg/mL、反应时间为4 h时酶的固定化效率可达50.5%,且保持77.3%相同含量酶的活性。对牛血清蛋白的酶解实验表明,在微波辅助作用下,磁性微球材料固定化酶可快速高效地酶解牛血清蛋白,酶解液在λ=280 nm处有较明显的吸收信号变化,HPLC分析表明酶解液中多肽片段吸收值变化明显,同时有新的多肽片段生成。

Fe3O4@mSiO2@nSiO2 can be modified with γ-aminopropyl triethoxysilane（APTES） for the immobilization of trypsin after glutaraldehyde cross-linking.The optimum conditions of the concentration of glutaraldehyde,the given amount of enzyme and reaction time are as following：1.875% glutaraldehyde（volume ratio）,0.3 mg/mL trypsin solution,4 h reaction time.Under these optimum conditions,the immobilizing rate and the activity retention rate are as high as 50.5% and 77.3% respectively.The immobilizing trypsin has a high efficiency on microwave assisted BSA digestion at 40 ℃.The results show that in hydrolyzate there is a variation peak absorb at λ=280 nm.A significant increasing absorbs of peptide fragments and some new peptide fragments can be observed by HPLC analysis.