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Michael addition of aldehydes to nitroolefins is an effective method to obtain useful chiral γ-nitroaldehydes. γ-Nitroaldehydes are precursors for chiral γ-aminobytiric acid analogues which have numerous pharmacological activities and are used for treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) has been developed and shown to be an effective biocatalyst for Michael addition of branched aldehydes to β-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine both containing the reactive ε-amino group, on the Michael addition catalyzed by 4-OT. Firstly, the effect of these mutations was examined by in silico analysis, followed by generation of three terminal ly-sine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for the ability to utilise β-nitrostyrene (1), (E)-2-(thiophene-2-yl)nitroethen (2) and p-chloro-trans-β-nitrostyrene (3) as Michael acceptors with isobutanal as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for the 4-OT catalysed Michael-additons, but it is not essential. [Projekat Ministarstva nauke Republike Srbije, br. 173048 i br. 172002]

Polyurethanes (PUs) are an exceedingly heterogeneous group of plastic polymers, widely used in a variety of industries from construction to medical implants. In the past decades, we have witnessed the accumulation of PU waste and its detrimental environmental impacts. PUs have been identified as one of the most toxic polymers leaching hazardous compounds derived both from the polymer itself and the additives used in production. Further environmental impact assessment, identification and characterization of substances derived from PU materials and establishing efficient degradation strategies are crucial. Thus, a selection of eight synthetic model compounds which represent partial PU hydrolysis products were synthesized and characterized both in terms of toxicity and suitability to be used as substrates for the identification of novel biocatalysts for PU biodegradation. Overall, the compounds exhibited low in vitro cytotoxicity against a healthy human fibroblast cell line and virtually no toxic effect on the nematode Caenorhabditis elegans up to 500 µg mL−1, and two of the substrates showed moderate aquatic ecotoxicity with EC50 values 53 µg mL−1 and 45 µg mL−1, respectively, on Aliivibrio fischeri. The compounds were successfully applied to study the mechanism of ester and urethane bond cleaving preference of known plastic-degrading enzymes and were used to single out a novel PU-degrading biocatalyst, Amycolatopsis mediterranei ISP5501, among 220 microbial strains. A. mediterranei ISP5501 can also degrade commercially available polyether and polyester PU materials, reducing the average molecular number of the polymer up to 13.5%. This study uncovered a biocatalyst capable of degrading different types of PUs and identified potential enzymes responsible as a key step in developing biotechnological process for PU waste treatment options. [ABSTRACT FROM AUTHOR]

Highlights: [•] Biocatalytic reduction of conjugated nitroalkenes is achieved using E. coli BL21(DE3). [•] Chemoselective biotransformations afforded product yields of up to 56%. [•] 2-(2-Nitropropyl)pyridine and 2-(2-nitropropyl)naphthalene synthetic route described. [•] E. coli BL21(DE3) has multiple active reductases able to biotransform nitroalkenes. [ABSTRACT FROM AUTHOR]