多孔陶瓷球固定化β-葡萄糖苷酶的研究

Immobilization of β-glucosidase on porous ceramic balls

以多孔陶瓷球为载体固定化β-葡萄糖苷酶,对固定化条件进行优化,确定陶瓷球固载β-葡萄糖苷酶的条件为:5 g陶瓷球载体与3 mL酶液,在4℃摇床中振荡交联反应2 h,低温干燥后,用含有0.3 g油性胶黏剂的溶液对固定化酶进行包封,酶液中β-葡萄糖苷酶浓度为36.5μmol/(mL.min),缓冲液pH为4.8,戊二醛体积分数0.5%。陶瓷球固定化β-葡萄糖苷酶的最适反应pH为4.8,游离酶pH为4.2,固定化和游离酶的最适反应温度均为60℃;固定化酶的酸碱、热和储存稳定性优于游离酶;固定化和游离酶的反应动力学常数Km分别为7.9 mmol/L和0.73 mmol/L;利用固定化酶重复分批酶解纤维二糖-葡萄糖混合糖液,前5批的酶解得率在80%以上。

With the objective of achieving practicable β-glucosidase immobilization,porous ceramic balls were taken as the carriers,and the conditions on β-glucosidase immobilization were studied.The optimum operation of β-glucosidase immobilization was as follows：5 g porous ceramic balls were put into 0.3 mL β-glucosidase solution（pH 4.8）,in which the concentrations of β-glucosidase and glutaraldehyde were 36.5 μmol/（mL·min） and 0.5%,respectively.The mixture was shaken at 4 ℃ for 2 h and then dried under low temperature.The immobilized β-glucosidase was obtained after enveloped by a solution which contained 0.3 g oil-based adhesive.The optimum reaction pH of immobilized β-glucosidase was 4.8,whereas the reaction pH of free β-glucosidase was 4.2.The optimum reaction temperatures of immobilized and free β-glucosidase were 60 ℃.Immobilized β-glucosidase offered the better properties than free β-glucosidase in pH,thermal and storage tests.The Km of immobilized and free β-glucosidase were 7.9 mmol/L and 0.73 mmol/L,respectively.A sugar mixture containing cellobiose and glucose was enzymatically hydrolyzed by the immobilized β-glucosidase.The yields of cellobiose conversion could be higher than 80% in 5 repeated batch operations.