632-20-2Relevant articles and documents
Single-Cell-Based Screening and Engineering of d -Amino Acid Amidohydrolases Using Artificial Amidophenol Substrates and Microbial Biosensors
An, Jung-Ung,Kim, Haseong,Kwon, Kil Koang,Lee, Dae-Hee,Lee, Hyewon,Lee, Jin-Young,Lee, Seung-Goo,Park, Sung Hyun,Rha, Eugene,Yeom, Soo-Jin
, p. 1203 - 1211 (2022/01/27)
Enantiomerically pure d-amino acids are important intermediates as chiral building blocks for peptidomimetics and semisynthetic antibiotics. Here, a transcriptional factor-based screening strategy was used for the rapid screening of d-stereospecific amino acid amidase via an enzyme-specific amidophenol substrate. We used a d-threonine amidophenyl derivative to produce 2-aminophenol that serves as a putative enzyme indicator in the presence of d-threonine amidases. Comparative analyses of known bacterial species indicated that several Bacillus strains produce amidase and form putative indicators in culture media. The estimated amidase was cloned and subjected to rapid directed evolution through biosensor cells. Consequently, we characterized the F119A mutation that significantly improved the catalytic activity toward d-alanine, d-threonine, and d-glutamate. Its beneficial effects were confirmed by higher conversions and recurrent applications of the mutant enzyme, compared to the wild-type. This study showed that rapid directed evolution with biosensors coupled to designed substrates is useful to develop biocatalytic processes.
Cβ-Selective Aldol Addition of d -Threonine Aldolase by Spatial Constraint of Aldehyde Binding
Park, Sung-Hyun,Seo, Hogyun,Seok, Jihye,Kim, Haseong,Kwon, Kil Koang,Yeom, Soo-Jin,Lee, Seung-Goo,Kim, Kyung-Jin
, p. 6892 - 6899 (2021/06/28)
d-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to β-hydroxy-α-d-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and Cβ-stereoselectivity in d-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 ? resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high Cβ-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTAG179A/S312A variant exhibits a near-perfect 99.5% de value for d-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.
Preparation and purification method of amino acid compound
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Paragraph 0066; 0067, (2018/06/21)
The invention relates to the field of industrial organic synthesis, in particular to a preparation and purification method of an amino acid compound. The method comprises the following steps that (1)alpha-amino nitrile compounds or hydantoin compounds or mixtures thereof are heated to react to obtain alpha-amino acid salt under the condition that volatile alkali and a suitable solvent exist; (2)after the alpha-amino acid salt obtained in step (1) is distilled, the alpha-amino acid salt is recrystallized in an organic solvent to obtain the alpha-amino acid compound. According to the method, reaction conditions are mild, materials can be recycled, and introduction of metal ions and use of ammonium carbonate salt are avoided, so that post-treatment is simple and no waste salt is generated.