通过重建底物结合口袋以提高其活性来工程化酮酸还原酶<a href="#fn1" title="提供电子补充信息（ESI）。参见DOI：10.1039 / c8cy02586j">†</a>,Catalysis Science & Technology

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通过重建底物结合口袋以提高其活性来工程化酮酸还原酶 †
Catalysis Science & Technology ( IF 4.4 ) Pub Date : 2019-03-19 00:00:00 , DOI: 10.1039/c8cy02586j
Di-Chen Wang _{1,

2,

3,

4,

5} , Heng Li _{1,

2,

3,

4,

5} , Shu-Ning Xia _{1,

2,

3,

4,

5} , Ya-Ping Xue _{1,

2,

3,

4,

5} , Yu-Guo Zheng _{1,

2,

3,

4,

5}

Affiliation

（R）-2-羟基-4-苯基丁酸（（R）-HPBA）是合成血管紧张素转化酶抑制剂的关键中间体。酮酸还原酶使2-氧代-4-苯基丁酸（OPBA）不对称还原代表了（R）-HPBA生产的一种有前途的方法，因为它具有高原子经济性和高达100％的理论收率。然而，酮酸还原酶的低活性限制了它们的工业应用。在这项研究中，这是首次设计具有优异活性的来自乳酸乳球菌的酮酸还原酶（Ll KAR），用于将OPBA还原为（R）-HPBA通过重建底物结合口袋。选择了几个残基（Leu127，Thr129，Leu244，Leu245和Ala250）进行诱变。通过三轮诱变，挑选出多个阳性突变体。与野生型L1 KAR相比，突变体L244G / A250G / L245R显示出比活性提高了3.5倍，催化效率（K _cat / K _m）提高了6.80倍。使用L244G / A250G / L245R结合西伯利亚克氏杆菌葡萄糖脱氢酶（GDH）进行辅因子再生，（R）-HPBA的生产率高达85.7 mM h ^-1反应体系中对映体过量> 99％。工程化的L244G / A250G / L245R代表了一种潜在的竞争性生物催化剂，可用于合成（R）-HPBA的实际应用。

"点击查看英文标题和摘要"

Engineering of a keto acid reductase through reconstructing the substrate binding pocket to improve its activity†

(R)-2-Hydroxy-4-phenylbutyric acid ((R)-HPBA) is a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. Asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by keto acid reductases represents a promising approach for (R)-HPBA production due to its high atom economy and theoretical yield of up to 100%. However, the low activity of keto acid reductases restricts their industrial application. In this study, it is the first time that a keto acid reductase from Leuconostoc lactis (LlKAR) with superior activity was designed for the reduction of OPBA to (R)-HPBA through reconstruction of the substrate binding pocket. Several residues (Leu127, Thr129, Leu244, Leu245, and Ala250) were selected for mutagenesis. Through three-round mutagenesis, multiple positive mutants were picked out. Compared with wild-type LlKAR, the mutant L244G/A250G/L245R exhibited 3.5-fold improvement in specific activity and 6.80-fold improvement in catalytic efficiency (K_cat/K_m). Using L244G/A250G/L245R coupled with Exiguobacterium sibiricum glucose dehydrogenase (GDH) for cofactor regeneration, the productivity of (R)-HPBA was up to 85.7 mM h⁻¹ with >99% enantiomeric excess in the reaction system. The engineered L244G/A250G/L245R represents a potential and competitive biocatalyst for practical application in the synthesis of (R)-HPBA.

更新日期：2019-03-19

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