壳聚糖交联介孔二氧化硅固定化β-葡萄糖苷酶生物转化淫羊藿苷研究  

Bioconversion of icariin by immobilizedβ-glucosidase in chitosan cross-linked mesoporous silica

作　　者：刘聪燕[1,2] 李瑞云[1,2] 马程遥 刘玉萍 瞿鼎[1,2] 李晓琦[1,2] 陈彦 LIU Cong-yan;LI Rui-yun;MA Cheng-yao;LIU Yu-ping;QU Ding;LI Xiao-qi;CHEN Yan(Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Multi-component of Traditional Chinese Medicine and Microecology Research Center,Jiangsu Academy of Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学附属中西医结合医院,江苏南京210028 [2]江苏省中医药研究院,中药组分与微生态研究中心,江苏南京210028

出　　处：《中草药》2021年第3期685-691,共7页Chinese Traditional and Herbal Drugs

基　　金：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(ZDRCA 2016036);江苏省中医药局科技项目(YB201925)。

摘　　要：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p H值为6.0,固定化时间为8h,酶质量浓度为7 mg/mL;固定化酶的酶活力为439.2μmol/(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10.24μg/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 immobilizedβ-glucosidase in chitosan cross-linked mesoporous silica,and improve the bioconversion of icariin to baohuoside I.Methods Theβ-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 immobilizedβ-glucosidase,and the enzyme leakage was also investigated.Using icariin as substrate and freeβ-glucosidase as control,the optimal enzymatic hydrolysis conditions,enzymatic kinetic parameters and recyclability of the immobilizedβ-glucosidase were investigated.Results To prepare the immobilizedβ-glucosidase,the optimal pH was confirmed to be 6.0,the optimal immobilization time was 8 h and the optimalβ-glucosidase concentration was 7 mg/mL.The immobilizedβ-glucosidase showed a well-retained activity of 439.2μmol/(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/m L,transformation time 8 h.The V_(max)and K_(m)of the immobilizedβ-glucosidase was 10.24μg/min and 16.15 mmol/L,respectively.After five cycles of reuse,the residual relative enzyme activity of the immobilizedβ-glucosidase was more than 80%.Conclusion The immobilizedβ-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.

关 键 词：淫羊藿苷 宝藿苷I Β-葡萄糖苷酶 介孔硅 固定化 生物转化 壳聚糖 吸附-交联法 N2吸附-脱附分析法 

分 类 号：R283.6[医药卫生—中药学]