海洋蔗糖磷酸化酶Suc75290催化合成α-熊果苷的条件优化及纯化工艺

Optimization of synthesis conditions and purification process ofα-arbutin catalyzed by marine sucrose phosphorylase Suc75290

摘要:

为探究来源于海洋的重组海洋蔗糖磷酸化酶Suc75290催化合成α-熊果苷的条件及其分离纯化条件，实验采用蔗糖和对苯二酚为底物，海洋蔗糖磷酸化酶Suc75290为催化剂合成α-熊果苷，使用大孔吸附树脂HP20对反应液中α-熊果苷和对苯二酚进行分离纯化。结果显示，pH为7的MES-NaOH缓冲液、反应温度为40 ℃、最适对苯二酚浓度为38.5 g/L、蔗糖与对苯二酚摩尔比最佳为5∶1（蔗糖浓度为600 g/L）、最适加酶量为300 U/mL、最适反应时间为28 h等6个因素是海洋蔗糖磷酸化酶Suc75290催化合成α-熊果苷的最优条件，对苯二酚的转化率为96.70%，α-熊果苷含量为64.3 g/L（238.7 mmol/L）；采用大孔树脂吸附法分离，以去离子水作为上样缓冲液，确定最佳分离条件：上样体积为10 mL，上样流速为0.5 mL/min，洗脱液为30%乙醇，洗脱流速1 mL/min，经过旋蒸处理后得到50 mg α-熊果苷，纯度为99.9%。综上，使用海洋蔗糖磷酸化酶Suc75290合成高浓度的α-熊果苷的条件优化及对α-熊果苷的分离纯化工艺研究为高纯度α-熊果苷的开发利用提供了应用基础。

Abstract:

In order to explore the conditions for the catalytic synthesis of α-arbutin by recombinant marine sucrose phosphorylase Suc75290 from the ocean and its separation and purification conditions， sucrose and hydroquinone were used as substrates， and marine sucrose phosphorylase Suc75290 was used as a catalyst to synthesize α-arbutin. The macroporous adsorption resin HP20 was used to separate and purify α-arbutin and hydroquinone in the reaction solution. The results showed that the optimum conditions for the synthesis of α-arbutin catalyzed by marine sucrose phosphorylase Suc75290 were as follows ： MES-NaOH buffer with pH value of 7， reaction temperature of 40°C， optimum hydroquinone concentration of 38.5 g/L， optimum molar ratio of sucrose to hydroquinone of 5∶1 （ sucrose concentration of 600 g/L）， optimum enzyme dosage of 300 U/mL， and optimum reaction time of 28 h. The conversion rate of hydroquinone was 96.70%， and the content of α-arbutin was 64.3 g/L （238.7 mmol/L）. The macroporous resin adsorption method was used for separation， and deionized water was used as the loading buffer. The optimal separation conditions were determined as follows： the loading volume was 10 mL， the loading flow rate was 0.5 mL/min， the eluent was 30% ethanol， and the elution flow rate was 1 mL/min. 50 mg of α-arbutin was obtained after rotary evaporation， and the purity was 99.9%. In summary， the optimization of the conditions for the synthesis of high-concentration α-arbutin using marine sucrose phosphorylase Suc75290 and the study on the separation and purification process of α-arbutin provide an application basis for the development and utilization of high-purity α-arbutin.