Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept
Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept
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DOI：
10.1002/cbic.200700500
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发表时间：
2007-12-17
期刊：
影响因子：
3.2
通讯作者：
Bommarius, Andreas S.
中科院分区：
文献类型：
--
作者：
Vazquez-Figueroa, Eduardo;Chaparro-Riggers, Javier;Bommarius, Andreas S.
在非天然加工条件下，特别是在高温下的不稳定性是阻止生物催化剂广泛用于工业合成的主要因素。用于合成手性化合物的许多氧化还原酶的关键区别是需要辅因子（例如，NAD（P）（H））功能。由于烟酰胺辅因子的价格高得令人望而却步，因此需要一种强有力的辅因子再生酶来通过生物催化经济地合成精细化学品。在这里，我们测试的结构指导的共识产生的热稳定的葡萄糖脱氢酶（GDH）。共有序列与另外的基于知识的标准组合用于选择用于置换的氨基酸。使用这种方法，我们产生了24种变体，其中11种表现出比野生型GDH更高的热稳定性，成功率为46%。在这24个变异体中，有7个位于亚基界面--已知会影响GDH稳定性--有6个更稳定（86%成功）。最好的变体的特征在于在65摄氏度下的半衰期类似于3.5天，而野生型在25摄氏度下的半衰期类似于20分钟，因此稳定性提高了10（6）倍。此外，三个最稳定的单突变转移到两个GDH同源物从苏云金芽孢杆菌和地衣芽孢杆菌。如通过GDH变体的半衰期和CD 222 nm测量的热稳定性增加，如预期的。由此产生的稳定性变化为以下观点提供了进一步的支持：这些残基对于GDH的稳定性至关重要，并加强了用于鉴定稳定突变的共识方法的成功。
Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing the widespread adoption of biocatalysts for industrial synthesis. A crucial distinction of many redox enzymes used to synthesize chiral compounds is the need for cofactors (e.g., NAD(P)(H)) for function. Because of the prohibitively high prices of nicotinamide cofactors, a robust cofactor-regenerating enzyme is required for the economical synthesis of fine chemicals by biocatalysis. Here we test the structure-guided consensus for the generation of a thermostable glucose dehydrogenase (GDH). The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46%. Of the 24 variants, seven were located at the subunit interface-known to influence GDH stability-and six were more stable (86% success). The best variants feature a half-life of similar to 3.5 days at 65 degrees C, in contrast to similar to 20 min at 25 degrees C for the wild type, thus enhancing stability 10(6)-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologues from Bacillus thuringiensis and Bacillus licheniformis. The thermal stability as measured by half-life and CD222nm of the GDH variants was increased, as expected. The resulting stability changes provide further support for the view that these residues are critical for stability of GDHs and reinforce the success of the consensus approach for identifying stabilizing mutations.