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Issue 20, 2018
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In vitro evolution of an l-amino acid deaminase active on l-1-naphthylalanine

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L-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a promising biocatalyst for enantioselective biocatalysis that can be exploited to produce optically pure D-amino acids or α-keto acids. In this study, we improved the catalytic efficiency of PmaLAAD on L-1-naphthylalanine (L-1-Nal), a synthetic amino acid of biotechnological interest. Eight evolvable positions were identified by a molecular docking and evolutionary conservation analysis. These positions were subjected to site-saturation mutagenesis, producing “smaller but smarter” libraries of variants. The best variant (F318A/V412A/V438P PmaLAAD) possesses a ∼5-fold lower Km (0.17 mM) and a ∼7-fold higher catalytic efficiency (9.2 s−1 mM−1) on L-1-Nal than the wild-type enzyme. Molecular docking analysis suggests that the substitutions increase the active site volume, allowing better binding of the bulky L-1-Nal substrate. Bioconversion reactions showed that the F318A/V412A/V438P PmaLAAD variant outperforms the wild-type enzyme in the deracemization of D,L-1-Nal: the complete conversion of 0.6 mM of the L-enantiomer was achieved in about 15 min, which is ∼7.5-fold faster than the wild-type enzyme. In addition, the F318A/V412A/V438P PmaLAAD is efficiently employed, together with the M213G D-amino acid oxidase variant, to produce 1-naphtylpyruvate from racemic D,L-1-Nal in one pot.

Graphical abstract: In vitro evolution of an l-amino acid deaminase active on l-1-naphthylalanine

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Article information

03 Jul 2018
20 Sep 2018
First published
20 Sep 2018

Catal. Sci. Technol., 2018,8, 5359-5367
Article type

In vitro evolution of an L-amino acid deaminase active on L-1-naphthylalanine

R. Melis, E. Rosini, V. Pirillo, L. Pollegioni and G. Molla, Catal. Sci. Technol., 2018, 8, 5359
DOI: 10.1039/C8CY01380B

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