Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.

A homogenous transition metal catalysis that combines hydroformylation, hydrogenation and direct amination presents an elegant multi-step pathway for synthesising primary diamines from olefins and ammonia. The valuable intermediate TCDdiamine is obtained, which has a wide range of industrial applications as a monomer building block. The rhodiumcatalysed hydrohydroxymethylation step converts non-conjugated dienes to the intermediate diols. Ammonia is added in a second ruthenium-catalysed amination step to obtain primary diamines. The conditions for both reactions were first optimised independently and combined to design a tandem reaction. For the amination reaction of the diol, excellent diamine yields of up to 88 % in toluene were achieved. An analysis of the interactions between the two catalytic systems demonstrated that the conditions of both single reaction steps counteract each other, meaning the presence of either rhodium or ruthenium blocks the other respective reaction. Using a twostep approach, optimised reaction conditions were applied to achieve equally high diamine yields of 88 %.

Aldoxime synthesis directly starting from alkenes was successfully achieved through the combination of hydroformylation and subsequent condensation of the aldehyde intermediate with aqueous hydroxylamine in a one-pot process. The metal...