AUTHOR=Riedel Anika , Mehnert Marika , Paul Caroline E. , Westphal Adrie H. , van Berkel Willem J. H. , Tischler Dirk 

TITLE=Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

JOURNAL=Frontiers in Microbiology

VOLUME=6

YEAR=2015

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.01073

DOI=10.3389/fmicb.2015.01073

ISSN=1664-302X

ABSTRACT=<p>Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYE<italic>Ro</italic>2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium <italic>Rhodococcus opacus</italic> 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYE<italic>Ro</italic>2 was produced in <italic>Escherichia coli</italic> and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH<sub>2</sub> for the reduction of activated α,β-unsaturated alkenes. In the active form OYE<italic>Ro</italic>2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYE<italic>Ro</italic>2 showed highest specific activities (45-50 U mg<sup>-1</sup>) on maleimides, which are efficiently converted to the corresponding succinimides. The OYE<italic>Ro</italic>2-mediated reduction of prochiral alkenes afforded the (<italic>R</italic>)-products with excellent optical purity (<italic>ee</italic> &gt; 99%). OYE<italic>Ro</italic>2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYE<italic>Ro</italic>2 for application in industrial biocatalysis is discussed.</p>