Purification, Characterization, Molecular Cloning, and Expression of a New Aminoacylase fromStreptomyces mobaraensisThat Can HydrolyzeN-(Middle/Long)-chain-fatty-acyl-L-amino Acids as Well asN-Short-chain-acyl-L-amino acids

Abstract

We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

“…This regio-specificity of the enzyme encoded by SAM23877 1734 is surprising because the enzyme Sm-AA produced by S. mobaraensis was not able to catalyze N-acetyl-L-lysine hydrolysis [12]. This result supported the hypothesis that SAM23877 1734 likely encodes a second aminoacylase, different from the product of SAM23877 1485 and specific for the hydrolysis of N-α-acetyl-L-lysine.…”

“…Lipases are the most currently used enzymes to synthesize bioactive molecules, but their use involves the utilization of organic solvent as reaction medium. These enzymes are able to catalyze the acylation of amino acids or peptides with fatty acids in aqueous media [1,[8][9][10][11][12]. Studies reported the use of neoteric media such as ionic liquids, allowing the improvement of the solubility of peptides such as Lys-Ser in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim] + [PF6] − ) in comparison with 2-methyl-2-butanol and of the reaction performances [6].…”

“…This limits the acylation of polar molecules such as amino acids or peptides [5]. Sm-AA has the ability to hydrolyze amino acids acylated with long or short fatty chains (C 2 to C 16 ) [12]. However, depending on the peptide, the solubility can be still low and the viscosity of ionic liquids can lead to mass transfer limitations [7].…”

“…An alternative to lipases is the use of acylases. These enzymes are able to catalyze the acylation of amino acids or peptides with fatty acids in aqueous media [1,[8][9][10][11][12]. These enzymes are produced by various microorganisms including the filamentous bacteria from Streptomyces genus.…”

“…This enzyme has an optimal pH from 7 to 8 and an optimal temperature of 50°C. Sm-AA has the ability to hydrolyze amino acids acylated with long or short fatty chains (C 2 to C 16 ) [12]. The second enzyme is Sm-eLA (55 kDa monomer) which has been identified as a ε-lysine-acylase (Nε-acyl-L-lysine amidohydrolase).…”