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Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines
Uppsala University. (AlbaNova University Center)
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
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2020 (English)In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 362, no 4, p. 812-821Article in journal (Refereed) Published
Abstract [en]

A protein engineering approach for expanding the substrate scope of the (S)-selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl- and benzyl-substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2-diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)-1,2-diphenylethylamine with ee >99 % under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20 % (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzyl-substituted primary amines.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2020. Vol. 362, no 4, p. 812-821
Keywords [en]
Biocatalysis, Enzymes, Kinetic resolution, Aminotransferase, Protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-264573DOI: 10.1002/adsc.201901252Scopus ID: 2-s2.0-85076409853OAI: oai:DiVA.org:kth-264573DiVA, id: diva2:1374336
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QC 20191204

Available from: 2019-11-29 Created: 2019-11-29 Last updated: 2020-03-09

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Land, HenrikRuggieri, FedericaBerglund, Per
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