目的 制备壳聚糖交联SBA-15型介孔二氧化硅（SBA-15）固定化β-葡萄糖苷酶（β-Glc），促进淫羊藿苷高效转化为稀有宝藿苷I。方法 采用吸附-交联法将β-Glc固定在壳聚糖交联SBA-15上，以载酶量和相对酶活力为评价指标，对其固定化条件进行优化；采用透射电子显微镜（TEM）、扫描电子显微镜（SEM）、傅里叶变换红外光谱（FTIR）和N₂吸附-脱附分析法对固定化酶进行表征，并对其酶泄露行为进行考察；以淫羊藿苷为底物，以游离酶为对照，考察固定化酶的最适酶解条件、酶解动力学和重复利用性。结果 制备交联SBA-15固定化β-Glc的最佳pH值为6.0，固定化时间为8 h，酶质量浓度为7 mg/mL；固定化酶的酶活力为439.2 μmol/（h·g），载酶量为1.120 g/g载体，最适酶解条件为pH 6.0，转化温度50℃，底物质量浓度0.5 mg/mL，转化时间8 h，酶解动力学参数最大反应速率（V_max）为10.24 μg/min，米氏常数（K_m）为16.15 mmol/L，重复利用5次后残余酶活大于80%。结论 制备的交联SBA-15固定化β-Glc载酶量高、转化能力强、重复利用性好，可用于高效获取宝藿苷I。

Objective To prepare the immobilized β-glucosidase in chitosan cross-linked mesoporous silica, and improve the bioconversion of icariin to baohuoside I. Methods The β-glucosidase was immobilized in chitosan cross-linked mesoporous silica SBA-15 by adsorption-crosslinking method. The immobilization conditions were optimized using the enzyme loading capacity and relative enzyme activity as the test index. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and N₂ adsorption-desorption analysis were used to characterize the physicochemical properties of immobilized β-glucosidase, and the enzyme leakage was also investigated. Using icariin as substrate and free β-glucosidase as control, the optimal enzymatic hydrolysis conditions, enzymatic kinetic parameters and recyclability of the immobilized β-glucosidase were investigated. Results To prepare the immobilized β-glucosidase, the optimal pH was confirmed to be 6.0, the optimal immobilization time was 8 h and the optimal β-glucosidase concentration was 7 mg/mL. The immobilized β-glucosidase showed a well-retained activity of 439.2 μmol/(h·g) as well as a high enzyme loading capacity of 1.120 g/g support. The optimum hydrolysis conditions were as follows:pH 6.0, conversion temperature 50℃, substrate concentration 0.5 mg/mL, transformation time 8 h. The V_max and K_m of the immobilized β-glucosidase was 10.24 μg/min and 16.15 mmol/L, respectively. After five cycles of reuse, the residual relative enzyme activity of the immobilized β-glucosidase was more than 80%.Conclusion The immobilized β-glucosidase in chitosan cross-linked mesoporous silica has a high enzyme loading capacity, strong enzyme activity and good reusability, which is beneficial to the efficient production of baohuoside I.

R283.6

国家自然科学基金青年项目（81703714）；国家自然科学基金青年项目（81903858）；江苏省科教强卫医学重点人才项目（ZDRCA2016036）；江苏省中医药局科技项目（YB201925）