β-半乳糖苷酶能以乳糖为底物生产低聚半乳糖。利用前期构建的产β-半乳糖苷酶F441Y重组毕赤酵母为菌种,进行摇瓶发酵并诱导培养。酵母菌体经过离心、洗涤后作为全细胞催化剂用于转化乳糖制备低聚半乳糖。为了提高转化率,首先通过单因素实验,优化了反应的初始p H、温度、乳糖浓度、加酶量和反应时间等因素。在此基础上,采用正交实验进一步优化了反应条件,优化后的最佳条件为:反应初始p H 6.0,反应温度为80℃,乳糖浓度为80%(w/v),加酶量为5 U/m L。在最佳转化条件下,反应24 h,低聚半乳糖的最高转化率为59.9%。与游离酶转化方法相比,该反应体系中的杂蛋白和美拉德反应较少,更有利于低聚半乳糖产品的后续分离。该研究建立的全细胞酶转化方法,具有转化率高、简单易行以及容易操作等优点。

β-galactosidase can be employed to produce galacto-oligosaccharidess using lactose as the substrate.Previously,a recombinant P. pastoris strain containing the gene of β-galactosidase mutant F441 Y from Sulfolobus solfataricus was constructed in our lab. In this study,whole cells of recombinant P. pastoris were used as biocatalyst to produce galacto-oligosaccharides,and the conditions such as initial p H,temperature,lactose concentration,enzyme concentration and reaction time were optimized. The optimal conditions were as follows: 80%( w / v) lactose,p H6. 0,80℃,5U / m L enzyme. After reaction for 24 h,the yield of GOS reached 59. 9%. Compared with the GOS production process using free enzyme,the proposed transformation approach with whole cells exhibited less impurity proteins and Maillard reaction,which made it easier for the separation of GOS. This study laid a foundation for the application of β-galactosidase.