纤维素载体固定化β-葡萄糖苷酶及其在大豆异黄酮水解中的应用

摘要

This study was conducted to evaluate the feasibility of conversion of isoflavones in soymilk using cellulose-immobilized β-glucosidase. β-glucosidase was immobilized onto cellulose particles with a loading capacity of over 40 mg of enzyme/40 cm3carrier. The immobilized enzyme exhibited a significant efficiency for the conversion of 4-nitrophenyl-β-D-glucuronide(p-NPG)to p-nitrophenol.The optimal temperature for the enzyme was 50℃and the Michaelis constant (Km) value was determined to be (1.38 ± 0.20) mmol/L. All isoflavones in black soymilk were completely converted to deglycosylated derivatives within 30 min using 40 cm3of the immobilized enzyme as analyzed by high-performance liquid chromatography (HPLC). Experimental evaluation of the stability showed that immobilized β-glucosidase retained 60% of its original activity after 15 times repeated use and after 15 days of storage.%建立纤维素载体固定化β-葡萄糖苷酶系统,用于黑豆浆中大豆异黄酮去糖基化反应,并对该催化系统进行了可行性评价.结果表明,在40cm3纤维素颗粒上可结合超过40mg β-葡萄糖苷酶,并且能有效将4-硝基苯-β-D-葡糖苷酸水解成对硝基苯酚,其反应最适温度为50℃.经动力学测试后可得其米氏常数Km值为(1.38±0.20)mmol/L.将该固定化酶系统用于黑豆浆中大豆异黄酮去糖基化,通过高效液相色谱检测其转化效率,表明该系统可以在30min内利用40cm3含酶载体将50mL黑豆浆中的异黄酮全部转化为去糖基化形式.该固定化酶催化系统连续化催化稳定性实验表明,在经过15次反应以及15d之后,该固定化酶系统仍然可维持其60%的催化活性.