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首页> 外文期刊>Catalysis science & technology >Rational engineering of Acinetobacter tandoii glutamate dehydrogenase for asymmetric synthesis of l-homoalanine through biocatalytic cascades
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Rational engineering of Acinetobacter tandoii glutamate dehydrogenase for asymmetric synthesis of l-homoalanine through biocatalytic cascades

机译：理性的不动杆菌tandoii工程对不对称合成谷氨酸脱氢酶通过biocatalytic l-homoalanine的级联

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摘要

l-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase from Acinetobacter tandoii (AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity of AtGluDH towards alpha-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 M l-threonine, which is close to the solubility limit, we obtained a 99.9% yield of l-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L-1 h(-1) space-time yield. Additionally, the activities of the engineered AtGluDH towards some other hydrophobic amino acids were also improved to 1.1-11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
机译：l-Homoalanine,一个有用的构建块几种手性药物的合成通过biocascades使用天然合成氨基酸作为廉价的起始反应物。添加昂贵的外部代数余子式和的低效率亮氨酸脱氢酶向中间2-ketobutyric酸在工业应用中两个主要的挑战。在此,一个双重cofactor-dependent谷氨酸脱氢酶的不动杆菌tandoii(AtGluDH)被充分利用代数余子式的胞内池在使用全细胞催化。酶和之间的疏水网络终端衬底的甲基2-ketobutyric酸,严格的衬底特异性AtGluDH对alpha-ketoglutarate成功改变,活动获得的最有效突变体(K76L / T180C)是17.2倍的野生型蛋白。co-expression系统被成功构建为了帮助释放传质限制。接近溶解度的限制,我们获得了99.9%的收益率l-homoalanine只有3.5 h在不增加外部辅酶级联,给予99.9% ee和29.2 g l - 1 h(1)时空产量。工程AtGluDH向其他疏水氨基酸也提高到1.1 - -11.2倍。因此,一些双重的工程设计cofactor-dependent GluDHs不仅可以消除低催化活性的不自然基质还提高代数余子式这些酶的利用率工业应用。

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