Detection of arsenic-binding siderophores in arsenic-tolerating Actinobacteria by a modified CAS assay.

The metalloid arsenic is highly toxic to all forms of life, and in many countries decontamination of water and soil is still required. Some bacteria have mechanisms to detoxify arsenic and can live in its presence. Actinobacteria are well known for their ability to produce a myriad of biologically-active compounds. In the present study, we isolated arsenic-tolerant Actinobacteria from contaminated water in Saxony, Germany, and determined their ability to produce siderophores able to bind arsenic. The binding capacity of different siderophore-like compounds was determined by a modified chrome azurol S (As-mCAS) assay with As(III) at high pH and using CAS decolorization as a readout. Arsenic-tolerant isolates from three actinobacterial genera were identified by 16 S rRNA gene sequence analysis: Rhodococcus, Arthrobacter and Kocuria. The isolated Actinobacteria showed a high As(III)-binding activity by siderophore-like compounds, resulting in 82-100% CAS decolorization, as compared to the results with EDTA. The interaction between As(III) and siderophore-like compounds was also detected at neutral pH. In summary, our results suggest that the isolated arsenic-tolerant Actinobacteria produce siderophores that bind arsenic, and open new perspectives on potential candidates for decontaminating environments with arsenic and for other biotechnological applications.

Functional characterization and stability improvement of a 'thermophilic-like' ene-reductase from Rhodococcus opacus 1CP.

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of 'thermophilic-like' OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,ß-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg(-1)) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 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 OYERo2 for application in industrial biocatalysis is discussed.