Characterization of a Novel Methylaspartate Ammonia Lyase from E. coli O157:H7 for Efficient Asymmetric Synthesis of Unnatural Amino Acids,ACS Sustainable Chemistry & Engineering

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Characterization of a Novel Methylaspartate Ammonia Lyase from E. coli O157:H7 for Efficient Asymmetric Synthesis of Unnatural Amino Acids
ACS Sustainable Chemistry & Engineering ( IF 7.1 ) Pub Date : 2019-12-09 , DOI: 10.1021/acssuschemeng.9b05424
Zi-Fu Ni ₁ , Ying-Jie Zeng ₁ , Pei Xu ₁ , Ze-Wang Guo ₁ , Xiao-Yang Ou ₁ , Fei Peng ₁ , Ji-Guo Yang ₂ , Min-Hua Zong ₁ , Wen-Yong Lou _{1,

2}

Affiliation

The asymmetric addition of ammonia to unsaturated acids using engineered methylaspartate ammonia lyase (MAL) is a particularly attractive and atom-economic method for the synthesis of unnatural amino acids. However, owing to insufficient enzyme gene mining of MALs, the catalytic performances of MALs have only been characterized in a few organisms. Herein, we describe a novel MAL from Escherichia coli (E. coli) O157:H7, whose gene was derived from a genome mining strategy. The enzyme (designated as El-MAL) has been successfully expressed in E. coli BL21(DE3) and isolated and purified to homogeneity by using 6 × polyhistidine tag. El-MAL existed as a dimer in solution, consisting of two identical subunits (ca. 45 kDa). Enzymatic properties indicated that the enzyme performed maximum activity in the presence of Mg²⁺ at pH 8.5 and 25 °C. El-MAL accepted fumarate, mesaconate, maleate, citraconic acid, and itaconic acid as substrates in the amination reaction. To the best of our knowledge, such catalytic activity toward citraconic acid and itaconic acid has not been reported previously. Therefore, this novel MAL displayed with high stereoselectivity in an asymmetric amination reaction for the synthesis of unnatural amino acids may become a promising biocatalyst in further exploitation.

中文翻译：

从大肠杆菌O157：H7的新型甲基天冬氨酸氨裂解酶的表征，用于非天然氨基酸的有效不对称合成

使用工程改造的天冬氨酸甲酯氨裂合酶（MAL）将氨不对称加成至不饱和酸是合成非天然氨基酸的一种特别吸引人且原子经济的方法。然而，由于MALs的酶基因挖掘不足，MALs的催化性能仅在少数生物中被表征。在这里，我们描述了一种来自大肠杆菌（O.coli）O157：H7的新型MAL ，其基因来源于基因组挖掘策略。该酶（命名为El-MAL）已在大肠杆菌中成功表达BL21（DE3），并使用6×多组氨酸标签分离并纯化至均一。El-MAL在溶液中以二聚体形式存在，由两个相同的亚基（约45 kDa）组成。酶学性质表明，在pH 8.5和25°C的Mg ²⁺存在下，该酶具有最大的活性。El-MAL接受富马酸酯，间康酸酯，马来酸酯，柠康酸和衣康酸作为胺化反应的底物。据我们所知，这种对柠康酸和衣康酸的催化活性以前没有被报道过。因此，在不对称胺化反应中以高的立体选择性显示出的新型MAL用于合成非天然氨基酸可能会成为有前途的生物催化剂。

更新日期：2019-12-11

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