BOKU University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Biotechnol J. 2010 Aug;5(8):838-47. doi: 10.1002/biot.201000126.
A novel heterodimeric beta-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U(oNPG)/mg protein. The K(m), k(cat) and k(cat)/K(m) values for lactose and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 38 mM, 20 s(-1), 530 M(-1).s(-1) and 1.67 mM, 540 s(-1), 325 000 M(-1).s(-1), respectively. The temperature optimum of beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of beta-(1-->3) and beta-(1-->6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Gal, beta-D-Galp-(1-->3)-D-Gal, and the trisaccharides beta-D-Galp-(1-->3)-D-Lac, beta-D-Galp-(1-->6)-D-Lac.
从土壤分离的乳酸杆菌 KUB-ST10-1 的粗细胞提取物中，通过硫酸铵分级沉淀、疏水相互作用和亲和层析，纯化出一种分子量为 105 kDa 的新型异源二聚体β-半乳糖苷酶。电泳纯酶的比活为 97 U(oNPG)/mg 蛋白。乳糖和邻硝基苯-β-D-半乳糖吡喃糖苷(oNPG)的 K(m)、k(cat)和 k(cat)/K(m)值分别为 38 mM、20 s(-1)、530 M(-1).s(-1)和 1.67 mM、540 s(-1)、325 000 M(-1).s(-1)。β-半乳糖苷酶活性的最适温度为 60-65℃，测定 10 分钟，明显高于其他乳酸杆菌β-半乳糖苷酶的值。Mg(2+)离子显著增强了酶的活性和稳定性。L. pentosus β-半乳糖苷酶用于从乳糖生产益生元半乳糖低聚糖(GOS)。在 78%乳糖转化率下，总糖的 GOS 最大产量为 31%。该酶对β-(1-->3)和β-(1-->6)键的形成具有很强的偏好性，鉴定的主要转半乳糖基化产物为二糖β-D-Galp-(1-->6)-D-Glc、β-D-Galp-(1-->3)-D-Glc、β-D-Galp-(1-->6)-D-Gal、β-D-Galp-(1-->3)-D-Gal 和三糖β-D-Galp-(1-->3)-D-Lac、β-D-Galp-(1-->6)-D-Lac。
