Laboratorio de Higiene Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y Bromatología, Universidade de Santiago de Compostela , Lugo , Spain.
Departamento Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM) , Madrid , Spain.
Front Bioeng Biotechnol. 2016 Mar 7;4:21. doi: 10.3389/fbioe.2016.00021. eCollection 2016.
β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures.
米曲霉β-半乳糖苷酶通过转半乳糖基作用，为乳果糖（OsLu）衍生低聚糖的合成提供了高产率。聚合度（DP）≥3的低聚糖比二糖和四糖具有更高的体外双歧杆菌增殖作用。因此，在这项工作中，优化了影响 A.oryzaeβ-半乳糖苷酶合成 OsLu 特异性选择性的反应条件，以提高 DP≥3 的 OsLu 产量。还进行了用戊二醛-琼脂糖载体固定化β-半乳糖苷酶的实验，目的是使该过程具有成本效益和工业可行性。对于 DP≥3 的 OsLu 合成，可溶性和固定化酶的最佳条件分别为 50°C、pH6.5、450g/L 的乳果糖和 8U/mL 的酶，反应时间短，可获得约 50%（w/v）的总 OsLu 和约 20%（w/v）的 DP3 的 OsLu，6'-半乳糖基乳果糖是主要产物。然而，在 60°C、pH4.5、450g/L 的乳果糖和 8U/mL 的酶条件下，有利于二糖的选择性形成。固定化提高了酶对温度变化的稳定性，并允许重复使用酶。我们可以得出结论，在确定的最佳条件下，使用戊二醛-琼脂糖载体固定化的 A.oryzaeβ-半乳糖苷酶，是合成益生元 OsLu 混合物时使用可溶性β-半乳糖苷酶的有效且具有成本效益的替代方法。
