β-呋喃果糖苷酶的固定化及其在低聚乳果糖合成中的应用

Immobilization of β-fructofuranosidase for the synthesis of lactosucrose

摘要:

【目的】探索适宜的树脂作为载体固定β-呋喃果糖苷酶，并研究该固定化酶催化合成低聚乳果糖。【方法】选择9种大孔吸附树脂和碱性离子交换树脂固定β-呋喃果糖苷酶，筛选固定化效果较好的树脂作为载体。用聚乙烯亚胺(PEI)修饰得到PEI-树脂，采用吸附法将酶固定于PEI-树脂上，并对固定化条件进行优化。考察固定化酶的重复使用稳定性及其催化合成低聚乳果糖的能力。【结果】通过筛选发现大孔阴离子交换树脂D311固定化效果较好，经过PEI修饰后，D311固定化效果显著提高。用PEI修饰的载体PEI-D311固定果糖苷酶，最优固定化条件为：PEI浓度2%，加酶量103 U/g，吸附温度25 °C，吸附pH 6.0?8.0，吸附时间8 h。最优条件下固定化酶活达57 U/g，酶活回收率达55.3%。用固定化酶催化水解1 mol/L蔗糖，重复利用15批载体酶活没有明显降低。用固定化酶催化合成低聚乳果糖，8 h内低聚乳果糖产量最高达到137 g/L。【结论】PEI-D311固定的果糖苷酶具有较好的重复使用稳定性及较高的低聚乳果糖合成能力，这为固定化酶法生产低聚乳果糖研究奠定了基础。

Abstract:

[Objective] We explored an appropriate resin to immobilize β-fructofuranosidase (β-FFase) to produce lactosucrose. [Methods] β-FFase was immobilized onto nine adsorption and ion-exchange resin, to select the best resin for immobilization. To improve immobilization efficiency, β-FFase was immobilized onto the resin modified by polyethylenimine (PEI). A series of immobilization conditions were optimized. The stability of the immobilized enzyme and lactosucrose synthesized by the immobilized enzyme were also explored. [Results] Ion-exchange resin D311 was excellent for β-FFase immobilization. Immobilization efficiency was improved apparently when D311 resin activated by PEI. The immobilized conditions were as follows: 2% PEI, 103 U/g resin, 25 °C, pH between 6.0 and 8.0, 8 h adsorption time. Under these conditions, the immobilized enzyme activity reached 57 U/g, and immobilized yield was 55.3%. Using the immobilized β-FFase to hydrolysis 1 mol/L sucrose, the immobilized enzyme can be used for 15 times with no apparently activity loss, and approximately 137 g/L lactosucrose was synthesized within 8 h. [Conclusion] β-FFase immobilized onto the resin modified by PEI showed a good operational stability and a high productivity of lactosucrose.