Biocatalytic potential of Ensifer adhaerens S-5 D-Carbamoylase: Insights from in silico and in vitro analyses.

Green, environmentally friendly technologies are currently emerging in respect of pharmaceutical and biotechnological industries' development. D-carbamoylase is a valuable catalyst for the D-amino acids industry since it catalyzes the second step of "hydantoinase process", the key step in the production of food ingredients, drug precursors and drug intermediates. We conducted in silico and in vitro analyses to characterize D-carbamoylase from Ensifer adhaerens S-5, a synthetic gene that was cloned and expressed in Escherichia coli. The in silico study included generating a three-dimensional model of the enzyme using ColabFold, performing molecular docking to evaluate substrate binding affinities, and conducting 100 ns molecular dynamics (MD) simulations to observe the behavior of D-carbamoylase alone and in enzyme-ligand complexes. Overall, the MD simulations suggest thatthe enzyme alone and in complex with substrate remains stable during 100 ns simulation. Using molecular docking analysis, we examined substrates' affinityto the model and substrate-binding pocket interactions. Analysis showed that Val205 and Asp209 can form hydrogen bonds with N-carbamoyl-D-tryptophan. D-carbamoylase was overexpressed in Escherichia coli BL21 Star cells, with notable inclusion bodies formation. The inclusion bodies were treated with N-lauroylsarcosine to solubilize and refold the enzyme. Substrate specificity analysis revealed that the enzyme is active toward N-carbamoyl-D-tryptophan, N-carbamoyl-D-leucine, N-carbamoyl-D-valine, N-carbamoyl-D-phenylalanine, and N-carbamoyl-D-alanine. A parallel comparison was conducted between the studied Ensifer adhaerens carbamoylase and the enzyme previously characterized from Pseudomonas sp. strain KNK003A. The results encourage us to continue research and to explore the application of the enzyme in the "hydantoinase process" for production of D-amino acids.