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,β-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.
Siderophores play an important role in the solubilisation and mobilization of iron (III) and various metal ions. To have a useful method to test siderophores in culture supernatants for their metal binding affinity, we redesigned and optimized the liquid CAS-assay for selected metal ions. CAS-assay solutions were calibrated with desferrioxamine B in different concentrations to calculate DFOB-equivalents to get a semi-quantitative evaluation. With these assay solutions, we were able to test siderophores in culture supernatants for their ability to chelate with Fe, Al, Ga, Cu, V and As.
In the present study we explore the idea of biotechnologically produced metallophore mixtures as selective chelating compounds for economically valuable metals from various sources. A complex soil matrix with natural levels of metal mineralization was employed as a potential source of metals. We focused on gallium-chelating metallophore preparations of two soil bacteria (Gordonia rubripertincta CWB2 and Paracoccus denitrificans PD1222) which were compared to the commercially available desferrioxamine B (DFOB). As a reference, the binding of iron was analyzed. The herein described successful mobilization of metals such as gallium from soil provides first hints towards alternative strategies, such as phytomining, sensor development, or solvent extraction based on metallophores. The metallophore mixture produced by the strains showed best results at pH 8 and allowed to mobilize gallium about three times better as the pure commercially available DFOB.
Rhodococcus erythropolis S43 is an actinobacterium isolated from an arsenic-contaminated soil sample, collected from an old smelter site, including an arsenic smelter, in Germany. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of arsenic. Here, we present the microbiological features and genomic properties of this biotechnologically relevant strain. The 6,812,940 bp draft genome is arranged into 264 scaffolds of 848 contigs. It possesses 62.5% of CG content and comprises 6,040 coding sequences and 49 tRNA genes. Bioinformatic genome analysis showed the presence of arsenic-resistance genes. A complete ars operon was found containing the arsACDR cluster coding for ArsA (efflux pump ATPase), ArsC (arsenate reductase), ArsD (chaperone) and ArsR (ars operon regulator). Our results show that the arsC mRNA level significantly increased in response to arsenite and arsenate exposure, suggesting its involvement in the arsenic resistance phenotype of strain S43. In addition, this strain showed to have a plethora of genes coding for proteins involved in oxidative-stress response, including catalase, super-oxide dismutase, glutathione peroxidase-related genes, thioredoxin and thioredoxin reductase, suggesting it is highly tolerant to oxidative conditions. Finally, genes for radiation resistance, biodesulfurization, and oil and phenol degrading pathways were also detected. Altogether this data make R. erythropolis S43 a good candidate microorganism for bioremediation of highly contaminated environments and other industrial applications.
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