Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards parasubstituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.

Widely applicable background depletion step enables transaminase evolution through solid-phase screening / M. Planchestainer, E. Hegarty, C. Heckmann, L. Gourlay, F. Paradisi. - In: CHEMICAL SCIENCE. - ISSN 2041-6520. - 10:23(2019), pp. 5952-5958. [10.1039/C8SC05712E]

Widely applicable background depletion step enables transaminase evolution through solid-phase screening

L. Gourlay;
2019

Abstract

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards parasubstituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.
Settore BIO/10 - Biochimica
Settore AGR/13 - Chimica Agraria
2019
2019
Article (author)
File in questo prodotto:
File Dimensione Formato  
HEWT2019.pdf

accesso aperto

Tipologia: Post-print, accepted manuscript ecc. (versione accettata dall'editore)
Dimensione 647.19 kB
Formato Adobe PDF
647.19 kB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/643412
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 16
  • ???jsp.display-item.citation.isi??? 16
social impact