Jiyang College , Zhejiang Agriculture and Forestry University , Zhuji , Zhejiang 311800 , China.
J Agric Food Chem. 2018 Jul 25;66(29):7712-7721. doi: 10.1021/acs.jafc.8b02333. Epub 2018 Jul 16.
High-efficiency lactulose-producing enzyme of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (WT- CsCE) was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Conditions for enzyme aggregation and cross-linking were optimized, and a sugar-assisted strategy with less damage to enzyme secondary structures was developed to improve the activity yield of CLEAs up to approximately 65%. The resulting CLEAs with multiple-layer network structures exhibited an enlarged optimal temperature range (70-80 °C) and maintained higher activity at 50-90 °C. Besides, CLEAs retained more than 95% of their initial activity after 10 successive batches at 60 °C, demonstrating superior reusability. Moreover, CLEAs displayed an equivalent or higher catalytic ability to free WT- CsCE in lactulose biosynthesis, and the final sugar ratios were similar, lactulose 58.8-61.7%, epilactose 9.3-10.2%, and lactose 27.8-30%, with a constant isomerization selectivity of 0.84-0.87 regardless of enzymes used and temperature applied. The proposed strategy is the first trial for enzymatic synthesis of lactulose catalyzed by CLEAs of WT- CsCE.
热纤梭菌纤维二糖 2-差向异构酶（WT-CsCE）高效产乳糖酶以交联酶聚集体（CLEAs）的形式固定化。优化了酶聚集和交联的条件，并开发了一种糖辅助策略，对酶二级结构的损伤较小，可将 CLEAs 的活性产率提高到约 65%。具有多层网络结构的所得 CLEAs 表现出扩大的最佳温度范围（70-80°C），并且在 50-90°C 下保持更高的活性。此外，CLEAs 在 60°C 下连续 10 批次后仍保留超过初始活性的 95%，表现出卓越的可重复使用性。此外，CLEAs 在乳糖合成中表现出与游离 WT-CsCE 相当或更高的催化能力，最终糖比相似，乳糖 58.8-61.7%，表乳糖 9.3-10.2%，乳糖 27.8-30%，无论使用的酶和应用的温度如何，异构化选择性均保持恒定为 0.84-0.87。所提出的策略是首次尝试用 WT-CsCE 的 CLEAs 进行乳糖的酶促合成。
