Improving the performance of coupled racemase/acylase systems: new structural insights and novel tools for high-throughput screening

Guiomar Sanchez Carron, Dominic Campopiano, Toni Fleming

Research output: Contribution to journalMeeting abstractpeer-review

Abstract

A coupled enzymatic dynamic kinetic resolution (DKR) system to allow the preparation of synthetically useful amino acids has been developed and improved. We combined a previously engineered N-acetyl amino acid racemase (NAAAR G291D/F323Y) with an l- or d-specific acylase and used them in a preparative scale DKR to generate enantiomerically pure amino acids. In this work we describe the latest efforts to expand the synthetic utility of this process. We present two novel spectrophotometric assays to measure NAAAR activity towards different substrates, replacing the existing HPLC and In vivo selection assays. One of them links the NAAAR/acylase couple to an l-amino acid oxidase and peroxidase. The other uses a d-amino acid dehydrogenase with broad substrate specificity. These assays allow us to identify novel NAAAR substrates and facilitate the high-throughput screening of NAAAR saturation mutagenesis libraries. X-ray structural analysis of NAAAR mutants in complex with N-acetyl-d-naphthylalanine reveals active site residues involved in the accommodation of bulkier substrates. These assays, combined with structural insights, guide the engineering of new NAAARs with catalytic potential across a larger range of amino acids.
Original languageEnglish
Pages (from-to)S75
JournalNew Biotechnology
Volume31
Early online date10 Jul 2014
DOIs
Publication statusPublished - 25 Jul 2014

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