Abstract：
Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced β-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of β-galactosidase in immobilized cells towards o-nitrophenyl β-D-galactoside were determined to be 3.446 mM and 2210 μmol min-1 g-1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.
摘要：
低聚半乳糖（GOS），作为人乳寡糖的模拟物，是调节肠道菌群生态平衡的重要益生元。使用京尼平交联乳酸克鲁维酵母CGMCC2.1494建立了一种新型的无载体细胞固定化方法，该方法产生了β-半乳糖苷酶，GOS合成所必需的酶。所得固定化细胞通过热重分析表征为稳定的,并通过扫描电子显微镜分析(SEM)和傅里叶变换红外光谱(FTIR)证实为交联的。固定细胞中β-半乳糖苷酶对邻硝基苯基β-D-半乳糖苷的Km和Vmax值分别为3.446mM和2210μmolmin-1g-1。与游离细胞相比，固定化酶对热和有机溶剂的耐受性更高。随后将固定化的细胞用于使用植物来源的半乳糖作为底物的GOS合成。通过单因素实验和响应面法优化合成反应条件,导致49.1%的高收率。此外，固定化细胞表现出良好的可重用性，可重复用于至少20批GOS合成，酶活性在35℃保持在70%以上。
