Efficient Enzymatic Synthesis of Carbamates in Water Using Promiscuous Esterases/Acyltransferases.

Biocatalysis provides an attractive approach to facilitate synthetic reactions in aqueous media. Motivated by the discovery of promiscuous aminolysis activity of esterases, we exploited the esterase from Pyrobaculum calidifontis VA1 (PestE) for the synthesis of carbamates from different aliphatic, aromatic, and arylaliphatic amines and a set of carbonates such as dimethyl-, dibenzyl-, or diallyl carbonate. Thus, aniline and benzylamine derivatives, aliphatic and even secondary amines could be efficiently converted into the corresponding benzyloxycarbonyl (Cbz)- or allyloxycarbonyl (Alloc)-protected products in bulk water, with (isolated) yields of up to 99%.

Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.