State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao, 266237, P. R. China.
Microb Cell Fact. 2024 Nov 14;23(1):307. doi: 10.1186/s12934-024-02586-9.
2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing.
Here, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01% in the total product. However, the LrSP exhibited an initial forward reaction rate of 11.83/s and reached equilibrium of 56.90% at 110 h, indicating low glycerol affinity and conversion rate. To improve catalytic efficiency, the LrSP was overexpressed in Lb. paracasei BL-SP, of which the intracellular SPase activity increased by 6.67-fold compared with Lb. reuteri SDMCC050455. After chemically permeabilized with Triton X-100, the whole-cell biocatalysis of Lb. paracasei BL-SP was prepared and showed the highest activity, with the initial forward reaction rate improved to 50.17/s and conversion rate risen to 80.79% within 17 h. Using the whole-cell biocatalyst, the final yield of 2-αGG was 203.21 g/L from 1 M sucrose and 1 M glycerol.
The food grade strain Lb. paracasei was used for the first time as cell factory to recombinantly express the LrSP and construct a whole-cell biocatalyst for the production of 2-αGG. After condition optimization and cell permeabilization, the whole-cell biocatalyst exhibited 23.89% higher equilibrium conversion and 9.10-fold of productivity compared with the pure enzyme catalytic system. This work would provide a reference for large-scale bioprocess of 2-αGG.
2-O-α-D-葡萄糖基甘油（2-αGG）是化妆品、保健品和食品领域的一种有价值的成分。蔗糖磷酸化酶（SPase）是生物合成 2-αGG 的理想选择，但其葡萄糖基受体亲和力和热力学特性在很大程度上尚不清楚，限制了 2-αGG 的生产。
从乳杆菌和双歧杆菌中获得了三种 SPase，其中由乳杆菌 SDMCC050455 编码的 LrSP 具有最佳的转葡糖苷能力，产物中 2-αGG 占 86.01%。然而，LrSP 的初始正向反应速率为 11.83/s，在 110 h 时达到 56.90%的平衡，表明甘油亲和力和转化率低。为了提高催化效率，将 LrSP 在副干酪乳杆菌 BL-SP 中过表达，与乳杆菌 SDMCC050455 相比，细胞内 SPase 活性增加了 6.67 倍。用 Triton X-100 化学渗透化后，制备了副干酪乳杆菌 BL-SP 的全细胞生物催化剂，其初始正向反应速率提高到 50.17/s，17 h 内转化率提高到 80.79%。使用全细胞生物催化剂，从 1 M 蔗糖和 1 M 甘油中最终获得 203.21 g/L 的 2-αGG。
首次将食品级菌株副干酪乳杆菌用作细胞工厂，重组表达 LrSP，并构建全细胞生物催化剂生产 2-αGG。在条件优化和细胞渗透化后，与纯酶催化体系相比，全细胞生物催化剂的平衡转化率提高了 23.89%，产率提高了 9.10 倍。这项工作为 2-αGG 的大规模生物过程提供了参考。
