固定化α-L-鼠李糖苷酶水解朝藿定C制备淫羊藿苷研究

Preparation of Icariin by Hydrolyzing Epimedin C with Immobilizedα-L-Rhamnosidase

目的制备SBA-15型介孔二氧化硅固定化α-L-鼠李糖苷酶,促进朝藿定C高效转化为淫羊藿苷。方法通过氨基化和醛基化对SBA-15进行修饰,将α-L-鼠李糖苷酶共价偶联在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X射线衍射(XRD)、傅里叶变换红外光谱(FT-TR)、氮气吸附与脱附、扫描电镜(SEM)及透射电镜(TEM)等对固定化酶进行表征;以朝藿定C为底物,考察固定化酶的最适酶解条件、酶解动力学和重复利用性。结果制备固定化酶工艺的最佳pH值为3.5,温度为35℃,时间为4 h,酶质量浓度为8 mg·mL^(-1);固定化酶的酶活力为198.6μmol·h^(-1)·g^(-1),载酶量为256.9 mg·g^(-1),最适酶解条件为pH值4.5,转化温度50℃,底物质量浓度0.5 mg·mL^(-1),转化时间12 h,酶解动力学参数最大反应速率(Vmax)为0.505μg·min^(-1),米氏常数(K_(m))为0.787 mmol·L^(-1),循环利用4次后残余酶活在65%以上,具有良好的稳定性。结论制备的SBA-15固定化α-L-鼠李糖苷酶载酶量高、转化能力强、重复利用性好,可用于高效转化朝藿定C制备淫羊藿苷。

OBJECTIVE To prepare t he immobilizedα-L-rhamnosidase on SBA 15 mesoporous silica to promote the efficient conversion of epimedin C to icariin.METHODS SBA-15 was modified through amination and aldehydeylation,and theα-L-rhamnosidase was covalently coupled onto SBA-15.The immobilization conditions were optimized using the enzyme loading capacity and relative enzyme activity as evaluation index.X-ray diffraction(XRD),Fourier transform infrared spectroscopy(FT-IR),N2 adsorption-desorption analysis,scanning electron microscopy(SEM)and transmission electron microscope(TEM)were used to characterize the physicochemical properties of immobilizedα-L-rhamnosidase.Using epimedin C as substrate and freeα-L-rhamnosidase as control,the optimal enzymatic hydrolysis conditions,enzymatic kinetic parameters and recyclability of the immobilizedα-L-rhamnosidase were investigated.RESULTS The optimal pH was 3.5,the optimal temperature was 35℃,the optimal immobilization time was 4 h and the optimalα-L-rhamnosidase concentration was 8 mg·mL^(-1).The immobilizedα-L-rhamnosidase showed a well-retained activity of 198.6μmol·h-1·g^(-1)as well as a high enzyme loading capacity of 256.9 mg·g^(-1)support.The optimum hydrolysis conditions were as follows:pH 4.5,conversion temperature 50℃,substrate concentration 0.5 mg·mL^(-1),and transformation time 12 h.The Vmax and Km of the immobilizedα-L-rhamnosidase was 0.505μg·min^(-1)and 0.787 mmol·L^(-1),respectively.After four cycles of reuse,the residual relative enzyme activity of the immobilizedα-L-rhamnosidase was more than 65%,which showed good stability.CONCLUSION The immobilizedα-L-rhamnosidase has a high enzyme loading capacity,strong enzyme activity and good reusability,which can be used for efficient conversion of epimedin C to icariin.