Abstract
Ene-reductases originating from extremophiles are gaining importance in the field of biocatalysis due to higher-stability properties. The genome of the acidophilic iron-oxidizing bacterium “Ferrovum” sp. JA12 was found to harbor a thermophilic-like ene-reductase (FOYE-1). The foye-1 gene was ligated into a pET16bp expression vector system, and the enzyme was produced in Escherichia coli BL21 (DE3; pLysS) cells in yields of 10 mg L−1. FOYE-1 showed remarkable activity and rates on N-phenylmaleimide and N-phenyl-2-methylmaleimide (up to 89 U mg−1, >97 % conversion, 95 % (R)-selective) with both nicotinamide cofactors, NADPH and NADH. The catalytic efficiency with NADPH was 27 times higher compared to NADH. At the temperature maximum (50 °C) and pH optimum (6.5), activity was almost doubled to 160 U mg−1. These findings accomplish FOYE-1 for a valuable biocatalyst in the synthesis of succinimides. The appearance of a thermophilic-like ene-reductase in an acidic habitat is discussed with respect to its phylogenetic placement and to the genomic neighborhood of the encoding gene, awarding FOYE-1 a putative involvement in a quorum-sensing process.
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This project was financial supported by the Saxon Ministry of Science and Fine Arts and the European Union (EU) in the framework of the European Social Fund (ESF; project numbers 100,101,363 and 100,236,458).
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AS and SRU carried out the genome mining, cloning, and phylogenetic analysis. AS and DT carried out the recombinant protein production, purification, enzyme characterization (temperature and pH optimum, thermal stability), and kinetics (data acquisition and analysis). Substrate specificity, product analysis, and stereochemistry were established and analyzed by AS and CEP. AS, SUR, and DT drafted the manuscript. MM, MS, and CEP critically revised the manuscript. All authors read and approved the final manuscript.
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Scholtissek, A., Ullrich, S.R., Mühling, M. et al. A thermophilic-like ene-reductase originating from an acidophilic iron oxidizer. Appl Microbiol Biotechnol 101, 609–619 (2017). https://doi.org/10.1007/s00253-016-7782-3
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DOI: https://doi.org/10.1007/s00253-016-7782-3