Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in vitro Applications

Douglas Weber, D. Patsch, Annikka Neumann, Margit Winkler, Dörte Rother*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

Original languageEnglish
Number of pages12
JournalChemBioChem
Volume2021
Early online date2 Feb 2021
DOIs
Publication statusE-pub ahead of print - 2 Feb 2021

Keywords

  • aldehydes
  • carboxylate reductase
  • in vitro cofactor regeneration
  • inclusion bodies
  • soluble protein

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Biochemistry
  • Organic Chemistry

Fields of Expertise

  • Human- & Biotechnology

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