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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 microspheres immobilized snail enzyme and optimize the preparation process of ginseng rare saponin Compound K (CK) by microspheres immobilized with snail enzyme to transform ginsenoside Rb1 (Rb1). Methods The microspheres were prepared by cross-linking and embedding method, and the enzyme activity recovery was taken as the index. The optimum preparation process was optimized by orthogonal test. The optimal reaction temperature, the optimum reaction p H value, Thermostability, p H stability and storage stability. The single factor was used to investigate the effect of conversion temperature, substrate concentration, conversion time and the number of immobilized snail enzyme on the conversion rate, and optimize the preparation process. Results The optimal preparation conditions of immobilized snail enzyme were as follows: sodium alginate 2%, Ca Cl2 2%, Si O2 and snail enzyme 1: 1, Was 81.94%. Immobilized snail enzyme and free snail enzyme showed different properties in terms of thermal stability and p H value stability. The optimal reaction temperature of immobilized snail enzyme was 60 ℃, and the optimum reaction p H value was 5.0. Immobilized snail enzyme stored in 15 ℃ environment for 30 d, the enzyme activity recovery was 55.17%. The conditions for the preparation of ginseng rare saponin CK by immobilized snail enzyme were as follows: the conversion temperature was 55 ℃, the substrate concentration was 1.0 mg / m L, the transformation time was 36 h, the conversion was 5 times, the average conversion was 36.79% . Conclusion The immobilization of snail enzyme enhanced its stability and service life, and the conversion rate of ginseng rare saponin CK increased, the process was simple and suitable for industrial production.