Amino acid-based surfactants are skin-friendly biosurfactants with low inflammatory potential and biodegradability that are used in cosmetic formulations. Conventionally, N-acyl-L-amino acids are synthesized by the Schotten-Baumann reaction using fatty acyl chlorides. When aminoacylases are applied high conversions in biocatalytical approaches starting from free fatty acids are possible [1,2]. Bacterial aminoacylases capable of synthesis have been isolated from e.g. Mycolicibacterium [1], Paraburkholderia [2], and Streptomyces [3,4].

We identified and investigated two novel enzymes originating from S. griseus DSM 40236^T [5]. The genes were cloned and an α-aminoacylase (EC 3.5.1.14), designated SgAA, and an ε-lysine acylase (EC 3.5.1.17), designated SgELA, were recombinantly expressed in S. lividans TK23. A protocol for high cell density fermentation in bioreactors was established ensuring dispersed growth of the bacterial mycelium. Both aminoacylases were purified as Strep-tag II-fused proteins and biochemically characterized, focusing on its hydrolytic activity to determine temperature and pH optima and stabilities. The short-chain acyl aminoacylase SgAA hydrolyzed various acetyl amino acids at the N_α-position with a broad specificity regarding the amino acid moiety. Substrates with longer acyl chains, like lauroyl amino acids, were hydrolyzed to a lesser extent. The aminoacylase SgELA specific for the hydrolysis of N_ε-acetyl-L-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. While synthesis of acyl amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine in aqueous buffer, which reveals the enzyme"s potential for biocatalytic applications. Future work to optimize acylation conditions is intended.

[1] Haeger, G. et al., Microb Cell Fact 22, 77 (2023).

[2] Haeger, G. et al., App Microbiol Biotechnol 108, 93 (2024).

[3] Koreishi, M. et al., J Am Oil Chem Soc 82, 631-637 (2005).

[4] Koreishi, M. et al., J Biotechnol 141, 160–165 (2009).

[5] Haeger, G. et al., FEBS Open Bio, 13: 2224-2238 (2023).