橙皮素单葡萄糖苷的酶法生物合成与定向调控

Enzymatic biosynthesis and directional regulation of hesperetin-7-O-glucoside

采用高效液相色谱法(HPLC)测定橙皮苷酶的酶学性质,并探讨其催化橙皮苷转化为橙皮素单葡萄糖苷(hesperetin-7-O-glucoside,HMG)的反应历程与定向调控。结果表明:橙皮苷酶在20～70℃、pH 3. 0～7. 0具有较好的稳定性,且最适反应温度为60℃,最适反应pH值为4. 0;在最适反应温度和pH下,该酶的动力学常数K_m为296. 69μg/mL,最大反应速率V_m为0. 872 3μg/(mL·min);与HMG转化相关的关键酶α-L-鼠李糖苷酶的K_m为232. 58μg/mL,V_m为0. 397 9μg/(mL·min);在60℃、pH 4. 0,酶用量为15. 35 U、增溶剂添加量为0. 8%、葡萄糖添加量为3 mg/mL、酶解时间为3 h时,酶反应向有利于HMG转化的方向进行,此时HMG得率为35. 8%。

Hesperetin-7-O-glucoside(HMG)is a precursor substance of a new sweetener.The enzymatic properties of hesperidinase were determined by high performance liquid chromatography(HPLC).The reaction mechanism and directional regulation of the conversion of hesperidin to HMG were discussed.Results showed that hesperidinase had good stability at 20-70℃and pH 3.0-7.0,the optimum reaction temperature and pH was 60℃and 4.0,respectively.Furthermore,at the optimum reaction temperature and pH,the kinetic constant K m of hesperidinase was 296.69μg/mL,the maximum reaction rate V m was 0.872 3μg/(mL·min).Also,the K m ofα-L-rhamnosidase related to HMG transformation was 232.58μg/mL and the V m was 0.397 9μg/(mL·min).The optimal hydrolysis conditions were pH 4.0 and hydrolysis temperature of 60℃,enzyme dosage 15.35 U,the amount of solubilizer 0.8%,the amount of glucose 3 mg/mL,and hydrolysis time 3 h.The enzymatic reaction was carried out using these conditions in favor of HMG transformation,and the yield of HMG was 35.8%.