Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.
Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.
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DOI：
10.1186/s12934-020-01501-2
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发表时间：
2021-01-06
影响因子：
6.4
通讯作者：
Fang B
中科院分区：
文献类型：
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作者：
Liao L;Zhang Y;Wang Y;Fu Y;Zhang A;Qiu R;Yang S;Fang B
l-叔亮氨酸是一种重要的医药中间体，利用辅助因子再生系统友好高效地合成l-叔亮氨酸一直是值得研究的课题。亮氨酸脱氢酶（leucine dehydrogenase, LeuDH）和葡萄糖脱氢酶（glucose dehydrogenase， GDH）的辅助因子再生体系在l-tle的合成中表现出很强的偶联催化效率，但GDH和LeuDH的多酶复合物尚未成功构建。本研究构建了一种新的融合酶（GDH - r3 - LeuDH），通过刚性肽连接剂介导LeuDH和GDH的融合，用于l-tle的高效生物合成。与游离酶相比，融合后的GDH-R3-LeuDH的环境耐受性和热稳定性均有较大提高。这种融合结构也加快了辅因子的再生速度，保持了酶的活性，因此GDH-R3-LeuDH的l-tle的产量和产量都提高了两倍。最后，在最佳催化条件（pH 9.0, 30°C, 0.4 mM NAD+， 500 mM底物包括三甲基丙酮酸和葡萄糖）下，在200 mL规模体系中，GDH-R3-LeuDH全细胞催化L -tle的空时产率可达到2136 g/L/d。这是迄今为止首次报道GDH和LeuDH作为多酶复合物融合合成l-tle并达到最高时空产率。这些结果证明了GDH-R3-LeuDH融合酶在高效生物合成l-tle方面的巨大潜力。
Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.
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影响因子：
--
作者：
Agosta, Eleonora;Caligiuri, Antonio;Canevotti, Renato
通讯作者：
Canevotti, Renato
影响因子：
2.8
作者：
Hoelsch, Kathrin;Weuster-Botz, Dirk
通讯作者：
Weuster-Botz, Dirk
影响因子：
--
作者：
Hong, Eun Young;Cha, Minho;Kim, Byung-Gee
通讯作者：
Kim, Byung-Gee
亮氨酸脱氢酶和甲酸脱氢酶双功能酶级联的开发提高了L-叔亮氨酸的生物合成
影响因子：
3
作者：
Lu, Jixue;Zhang, Yonghui;Fang, Baishan
通讯作者：
Fang, Baishan
影响因子：
3.9
作者：
Böttcher T
通讯作者：
Böttcher T