微球固定化蜗牛酶转化人参皂苷Rb1制备人参稀有皂苷Compound K研究

Transformation of rare ginsenoside Compound K from ginsenoside Rb_1 catalyzed by snailase immobilization onto microspheres

目的制备微球固定化蜗牛酶,并优选出微球固定化蜗牛酶转化人参皂苷Rb1(Rb1)制备人参稀有皂苷Compound K(CK)的最佳制备工艺。方法采用交联-包埋法制备微球固定化蜗牛酶,以酶活力回收率为考察指标,通过正交试验优选出最佳制备工艺;考察了最适反应温度、最适反应p H值、热稳定性、p H稳定性和储存稳定性等酶学性质,并通过单因素考察转化温度、底物浓度、转化时间和固定化蜗牛酶使用次数对转化率的影响,优化制备工艺。结果固定化蜗牛酶的最佳制备工艺:海藻酸钠质量浓度2%,Ca Cl2质量浓度2%,Si O2与蜗牛酶质量比1∶1,在此条件下固定化蜗牛酶的酶活回收率为81.94%;固定化蜗牛酶与游离蜗牛酶在热稳定性与p H值稳定性方面显示出不同的性质,其中固定化蜗牛酶的最适反应温度为60℃,最适反应p H值为5.0。固定化蜗牛酶在15℃环境下保存30 d后,酶活回收率为55.17%。固定化蜗牛酶转化Rb1制备人参稀有皂苷CK的转化条件:转化温度为55℃,底物质量浓度为1.0 mg/m L,转化时间为36 h,转化次数为5次,平均转化率为36.79%。结论蜗牛酶的固定化增强了其稳定性和使用寿命,且人参稀有皂苷CK的转化率提高,工艺简单,适合工业化生产。

Objective To prepare snailase immobilization onto microspheres and to optimize the process conditions for the transformation of rare ginsenoside Compound K （CK） from ginsenoside Rbl （Rbl） catalyzed by snailase immobilization onto microspheres. Methods Considering the recovery rate of enzyme activity as the target, crosslink-embedding method was used for preparing the snailase immobilization onto microspheres and optimizing the preparation technology by orthogonal test. Furthermore, the enzyme characterization of temperature, enzymatic properties of pH value, thermal stability, pH stability, and storage stability was studied, and the effectiveness of temperature, concentration reaction time, and transformational times on the bioconversion rate was studied to optimize the preparation conditions. Results The best process was achieved at 2% sodium alginate, 2% CaC12, SiO2 and snail enzyme mass ratio of 1 ： 1, with the above conditions, the enzyme activity recovery rate was 81.94%, immobilization snailase and free snailase exhibit different properties about thermal stability and pH stability, the optimum temperature was 60 ~C, and the optimum pH was 5.0. Under these conditions, the snailase immobilization onto microspheres remained 55. l 7% enzyme activity when storaged at 15 ~C for 30 d. The best process was achieved at 55 ~C, the substrate concentration was 1.0 mg/mL, the conversion time was 36 h, the effective continuous transformational times were five rounds and the average transformational ratio for rare ginsenoside CK was up to 36.79%. Conclusion The results concluded from the experiments indicate that the immobilization procedure could promote theresistance of enzyme against temperature, pH shift, and some other tough reaction conditions, meanwhile prolong the enzymatic lifetime for storage. The bioconversion rate is impoved and it is feasible to prepare rare ginsenoside CK by enzymolysis with snailase immobilization onto microspheres. Besides, the condition is moderate and it is suitable for industrialization.