Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10–His216 and His216–Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Natural ectoine, (+)-(4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, is an extremely important small biomolecule belonging to the class of osmolytic/osmoprotective compounds. It stabilizes biomacromolecules such as DNA and proteins and protects them from denaturation by heat, dehydration, and UV radiation. The rapidly growing interest in this compound resulted in currently exclusive biotechnological production, while a chemical process along with enantioseparation as an alternative has not yet been established. An improved chemical synthesis of racemic ectoine starting from γ-butyrolactone in very good yield is described. Regioselective monoacetylation is achieved by the complexation of a copper(II)-ion with two molecules of 2,4-diamonobutyric acid in the key synthetic step. The racemic ectoine was synthesized with the aim of being successfully enantioseparated for the first time by high-performance liquid chromatography (HPLC) using a teicoplanin-based Chiral-T column in different solvent systems. The presence of (+)-ectoine was determined and quantified using an HPLC protocol on the Synergy Polar-RP column in fermentation broths inoculated with different strains of Streptomyces sp. bacteria isolated from the Adriatic Sea and grown on different NaCl concentrations.