Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus.
Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus.
复制标题
DOI：
10.1186/s12934-017-0841-3
复制
发表时间：
2017-12-12
影响因子：
6.4
通讯作者：
Lee HC
中科院分区：
文献类型：
--
作者：
Park AR;Kim JS;Jang SW;Park YG;Koo BS;Lee HC
乳果糖是一种人工合成的双糖，由于其作为益生元的作用，特别是增殖双歧杆菌和乳杆菌，并增强钙和镁的吸收，已受到越来越多的关注。纤维二糖2-差向异构酶（CE）的使用被认为是乳果糖工业生产的一种令人感兴趣的替代方案。CE在未修饰的β-1，4-连接寡糖（包括β-1，4-甘露二糖、纤维二糖和乳糖）的还原端将d-葡萄糖残基可逆转化为d-甘露糖残基。最近，一些CE三维结构的报道，揭示了机制的细节。利用这些信息，我们重新设计了CE的底物结合位点，将其活性从差向异构化扩展到异构化。使用3个已知的CE结构模型的叠加，我们确定了2个残基（Tyr 114，Asn 184），似乎在结合epilactose中发挥重要作用。我们修改了这些残基，其与甘露糖部分的C2相互作用，以防止差向异构化为epilactose。我们发现Y114 E突变导致65 °C下副产物乳果糖的释放增加，而其活性在37 °C下较低。值得注意的是，这种现象仅在高温下观察到，并且当基底增加时更可靠。使用Y114 E，在优化的条件下研究了乳糖异构化为乳果糖，当使用200 g/l乳糖时，2 h得到86.9 g/l乳果糖和4.6 g/l表乳糖。这些结果表明，Y114 E突变增加了乳糖的异构化，同时降低了乳糖的差向异构化。因此，活性位点口袋的细微修饰可以将其天然活性从差向异构化扩展到异构化，而不会显著损害底物结合。虽然还需要进一步的研究来将其扩展到工业过程，但我们证明了基于结构分析的工程化这种酶的潜力。本文的在线版本（10.1186/s12934-017-0841-3）包含补充材料，可供授权用户使用。
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. The online version of this article (10.1186/s12934-017-0841-3) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
8.8
作者：
Hua, Xiao;Yang, Ruijin;Zhao, Wei
通讯作者：
Zhao, Wei
影响因子：
3.1
作者：
Khatami, Seyyedhadi;Ashtiani, Farzin Zokaee;Mehrdad, Mahsa
通讯作者：
Mehrdad, Mahsa
影响因子：
--
作者：
Park, Ah-Reum;Koo, Bong-Seong;Lee, Hyeon-Cheol
通讯作者：
Lee, Hyeon-Cheol
影响因子：
1.6
作者：
Senoura, Takeshi;Taguchi, Hidenori;Ito, Susumu
通讯作者：
Ito, Susumu
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH