重组β-半乳糖苷酶的制备及转化条件优化

摘要

以前期构建的产Bacilluscirculansβ-半乳糖苷酶重组大肠杆菌E.coli BL21 (DE3)/pET-20b-lac为菌种,进行摇瓶发酵诱导培养,培养24 h胞外上清酶活迭15 U/mL.利用制备的β-半乳糖苷酶粗酶液进行酶转化实验.优化了酶转化条件,考查了初始pH、反应温度、乳糖质量浓度、加酶量和反应时间等因素对低聚半乳糖产率的影响.确定最优转化条件为:初始pH 6.5、反应温度55℃,起始乳糖质量浓度为700 g/L,加酶量为8 U/mL.在此条件下反应16h,低聚半乳糖转化率可迭57％.%Previously,a recombinant straincontaining the encoding gene of Bacillus circulans β-galactosidase,E.coli BL21 (DE3)/pET-20b-lac,was constructed in our laboratory.In this study,the strain was cultured in shake flask for 24 h,reaching an extracellular enzyme activity of 15 U/mL.The crude enzyme was used for the preparation of oligosaccharides.The reaction conditions,including initial pH,reaction temperature,lactose concentration,enzyme dosage and reaction time,forthe yield ofgalactooligosaccharides was optimized.The optimal conditions for the conversion was as the following:initial pH 6.5,reaction temperature 55 ℃,initial lactose concentration 700 g/L,enzyme concentration 8 U/mL.When the reaction was performed under this specified conditions,the galactooligosaccharide yield reached the maximum of 57％ at 16 h.