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Issue 6, 2019
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Improving the catalytic efficiency and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 by semi-rational engineering en route to chiral γ-amino acids

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Abstract

Nitrilase-catalysed desymmetrization of 3-substituted glutaronitriles to optically active 3-substituted 4-cyanobutanoic acid offers an attractive approach to access chiral β-substituted γ-amino acids, an important moiety in pharmaceuticals. In this study, we employed enzyme-substrate docking and alanine scanning to determine the key amino acid residues which have a positive effect on the activity and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 (SsNIT). Then, site-saturation mutagenesis and combinatorial mutagenesis of the positive amino acid residues (H141, P194, M197, I201 and F202) were performed, and a double mutant (P194A/F202V) and a triple mutant (P194A/I201A/F202V) with high activity and stereoselectivity were identified for desymmetrization of 3-substituted glutaronitriles to afford (S)-3-substituted-4-cyanobutanoic acids with a high space–time productivity of up to 488 g L−1 d−1. Molecular calculation suggests that the enzymatic activity improvement may be due to the enlargement of the substrate binding cavity. It is interesting that the stereoselectivity is enhanced simultaneously.

Graphical abstract: Improving the catalytic efficiency and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 by semi-rational engineering en route to chiral γ-amino acids

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Publication details

The article was received on 05 Dec 2018, accepted on 21 Feb 2019 and first published on 21 Feb 2019


Article type: Paper
DOI: 10.1039/C8CY02455C
Catal. Sci. Technol., 2019,9, 1504-1510

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    Improving the catalytic efficiency and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 by semi-rational engineering en route to chiral γ-amino acids

    S. Yu, P. Yao, J. Li, J. Feng, Q. Wu and D. Zhu, Catal. Sci. Technol., 2019, 9, 1504
    DOI: 10.1039/C8CY02455C

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