ForBioKorea Co., Ltd., Gasan digital 2-ro, Geumcheon-gu, Seoul, Republic of Korea.
Microb Cell Fact. 2017 Dec 12;16(1):224. doi: 10.1186/s12934-017-0841-3.
Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization.
Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used.
These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.
乳果糖是一种合成的二糖，由于其作为益生元的作用而受到越来越多的关注，特别是增殖双歧杆菌和乳酸杆菌，并增强钙和镁的吸收。使用纤维二糖 2-差向异构酶（CE）被认为是工业生产乳果糖的一种有趣的替代方法。CE 可逆地将 D-葡萄糖残基转化为未修饰的β-1,4-连接寡糖的还原端的 D-甘露糖残基，包括β-1,4-甘露二糖、纤维二糖和乳糖。最近，报道了几个 CE 的 3D 结构，揭示了其机制细节。利用这些信息，我们重新设计了 CE 的底物结合位点，使其活性从差向异构化扩展到异构化。
使用与 3 个已知 CE 结构模型的叠加，我们确定了 2 个残基（Tyr114、Asn184）似乎在结合表乳果糖中起着重要作用。我们修饰了这些与甘露糖部分的 C2 相互作用的残基，以防止向表乳果糖的差向异构化。我们发现 Y114E 突变导致在 65°C 时释放更多的副产物乳果糖，而在 37°C 时其活性较低。值得注意的是，这种现象仅在高温下观察到，并且在底物增加时更可靠。在优化条件下，使用 Y114E 研究了乳糖向乳果糖的异构化，当使用 200g/L 的乳糖时，2 小时内得到 86.9g/L 的乳果糖和 4.6g/L 的表乳果糖。
这些结果表明，Y114E 突变增加了乳糖的异构化，同时减少了乳糖的差向异构化。因此，对活性位点口袋的微妙修饰可以在不显著降低底物结合的情况下，将其天然活性从差向异构化扩展到异构化。虽然需要进一步的研究将其扩展到工业过程，但我们基于结构分析证明了对该酶进行工程改造的潜力。
