Abstract: In this work, experiments were explored to prepare the cross-linked enzyme aggregates (CLEAs) of β-glucosidase from black plum seeds and evaluate their stability. After determination of the optimal preparation parameters, including the optimum precipitation agent, volume ratio of precipitant to enzyme liquid and glutaraldehyde concentration, then the shape and surface morphology of the CLEAs were examined using scanning electron microscopy. Moreover, the salidroside synthesis catalyzed by CLEAs was chosen as a model reaction to investigate the thermal and operational stabilities. The optimum precipitation agent, volume ratio of precipitant to enzyme liquid, glutaraldehyde concentration and crossing-linking time were found to be 95% ethanol, 1:3 (v:v), 30 mmol·L^-1 and 1.0 h, under which the activity recovery of CLEAs recorded 91.4%. Compared to free enzyme, the surface morphology of CLEAs displayed a very high specific surface area, abundant pores and dense channels. Experimental data of the thermal stability indicated that CLEAs retained 75.1% of their original activity after incubation for 2.0 h at 60℃, which was 6.1 times high than that of the free enzyme. For the operational stability, 73.5% of the initial activity of CLEAs was still maintained after eight successive cycles of reuse. This study proves CLEAs of β-glucosidase from black plum seeds exhibited good activity, thermal stability and operational stability, which was advantageous to the synthesis of the glycosides.

Key words: black plum seeds, β-glucosidase, cross-linked enzyme aggregates, glycoside, stability

摘要： 为探究β-葡萄糖苷酶聚合物的制备方法及其稳定性,以黑布林籽β-葡萄糖苷酶为原料制备交联酶聚合物(CLEAs),优化了制备过程中沉降剂的种类及比例、戊二醛交联剂的浓度等工艺参数,并采用扫描电镜分析其表面结构特征;在此基础上,以红景天苷的酶促合成为模型反应,研究CLEAs在有机溶剂中的热稳定性和操作稳定性。结果表明,制备β-葡萄糖苷酶CLEAs的最佳沉降剂为95%乙醇,其适宜添加量为酶液体积的3倍;戊二醛交联剂的最适浓度为30 mmol·L^-1,交联时间为1.0 h,酶活回收率高达91.4%。扫描电镜结果表明,在此条件下制备的CLEAs有较大的比表面积和致密的多孔道结构。稳定性结果表明,将β-葡萄糖苷酶CLEAs在60℃下保温2.0 h,其酶活回收率为75.1%,是游离酶的6.1倍;重复使用8次后,仍保持73.5%的初始酶活性。综上,黑布林籽β-葡萄糖苷酶CLEAs具有良好的生物催化活性、热稳定性和操作稳定性,可用于糖苷类化合物的高效合成。本研究为β-葡萄糖苷酶CLEAs的研究提供了理论依据,对工业化生产具有现实意义。

关键词: 黑布林籽, β, -葡萄糖苷酶, 交联酶聚合物, 糖苷, 稳定性