School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Molecules. 2021 Feb 25;26(5):1248. doi: 10.3390/molecules26051248.
α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.
α-半乳糖寡糖（α-GOSs）具有作为益生元和治疗剂的重要功能。本工作首次建立了固定化α-半乳糖苷酶分批合成α-GOSs 的方法，为未来的工业应用奠定了基础。通过酶沉淀和交联步骤，无需使用载体，将 L63 来源的α-半乳糖苷酶固定化为交联酶聚集体（CLEAs）纳米生物催化剂。在测试的试剂中，硫酸铵表现出高沉淀效率，并通过扫描电子显微镜和傅里叶变换红外光谱分析诱导α-半乳糖苷酶 CLEAs（Aga-CLEAs）的规则结构。通过响应面法优化，在 0.55 U/mL 酶和 36.43 mM 戊二醛交联 1.71 h 的条件下，硫酸铵诱导的 Aga-CLEAs 的酶活回收率约为 90%。Aga-CLEAs 表现出更高的热稳定性和有机溶剂耐受性。储存能力也得到了提高，因为它在 4°C 下储存三个月后仍保持 74.5%的活性，明显高于游离酶（21.6%）。此外，Aga-CLEAs 在从半乳糖合成 α-GOSs 中的重复使用性能优异，在 10 次分批反应后保留了超过 66%的酶活，产物收率均高于 30%。
