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Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in vitro Applications
Author(s) -
Weber Douglas,
Patsch David,
Neumann Annika,
Winkler Margit,
Rother Dörte
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000846
Subject(s) - carboxylate , heterologous , benzaldehyde , heterologous expression , biochemistry , enzyme , chemistry , escherichia coli , combinatorial chemistry , biocatalysis , reductase , stereochemistry , catalysis , recombinant dna , reaction mechanism , gene
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 ( No CAR) 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 No CAR. This highly active No CAR 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.