Molecular rules for selectivity in lipase-catalysed acylation of lysine

International audienceThe selectivity of L-lysine acylation by lauric acid catalysed by Candida antarctica lipase B (CALB) was investigated combining experimental and theoretical methodologies. Experiments showed the near-exclusive acylation of lysine ε-amino group; only traces of product resulting from the acylation of lysine α-amino group were observed fleetingly. Molecular modelling simulations were performed aiming to understand the molecular rules for selectivity. Flexible docking simulations combined with structural investigations into lysine/CALB binding modes also suggested the preferential acylation of lysine ε-amino group without, however, excluding the acylation of the lysine α-amino group. Electrostatic interaction energy between lysine and the residues covering the catalytic cavity was calculated in order to understand the discrimination between the two lysine amino groups. The results suggests that the proximity of the carboxylate group hinders the binding of the substrate in configurations enabling the N-acylation. Key interactions with the polar region covering the catalytic triad were identified and a plausible explanation for selectivity was proposed

N-α-acylation of lysine catalyzed by immobilized aminoacylases from Streptomyces ambofaciens in aqueous medium

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Prediction of short peptides composition by RP-HPLC coupled to ESI mass spectrometry

International audienceThe combination of reversed-phase chromatography and electrospray mass spectrometry was used to predict the amino acid composition of low molar weight peptides present in a complex mixture of rapeseed protein hydrolysates. First, amino acid retention times on a hydrophobic stationary phase were evaluated to determine hydrophobicity coefficients for each amino acid and then global hydrophobicity coefficients were established for standard peptides. In this way, a correlation between peptide hydrophobicity coefficients and retention time was established. Then, a C language program was developed to calculate all potential amino acid combinations from the mass of a peptide (molar mass < 1000 Da) and determine theoretical hydrophobicity coefficients corresponding to these combinations. Comparison between the theoretical retention time determined by the model and the experimental retention time resulted in the establishment of a sorted potential composition table. This methodology has been applied to two different rapeseed protein hydrolysates peptides that have been purified and sequenced

Prediction of the amino acid composition of small peptides contained in a plant protein hydrolysate by LC–MS and CE–MS

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Enzymatic acylation of the polar dipeptide, carnosine: Reaction performances in organic and aqueous media

IATE Axe 4 : Biotechnologie microbienne et enzymatique des lipides et des agropolymères Contact: [email protected] audienceThe major limitation of lipase-catalyzed acylation of the polar bioactive dipeptide carnosine (β-Ala-His) is its very low solubility in solvents other than water. In the current work, two approaches were developed to overcome this constraint. The first acylation strategy consisted of the common use of a lipase in an anhydrous organic medium. However, the peptide is only weakly soluble in an anhydrous organic medium. A preliminary step of high pressure homogenisation was performed to improve the dispersion of carnosine particles and to reduce the particle size, thereby enhancing the accessible area of the substrate to the enzyme. The second approach used an aqueous medium, in which carnosine was totally solubilized, and aimed to evaluate the ability of an acyltransferase to catalyze acylation of the peptide. The enzymatic acylation of carnosine in anhydrous organic solvent was catalyzed by lipase B from Candida antarctica. The size of carnosine particles was decreased significantly in the organic solvent after high pressure pre-treatment and high temperature, leading to a substrate conversion yield of 39% after 120 h of reaction. The acyltransferase of Candida parapsilosis was demonstrated to be able to catalyze the acylation of carnosine in aqueous medium. Under these conditions, a substrate conversion yield of 48% was obtained when the reaction was run for 48 h, suggesting that this enzyme may be important to develop processes for acylation of polar peptides. In both reaction systems, the product was identified as N-acyl carnosine. The antioxidant activities of the purified amide were determined and compared to those of carnosine. Its xanthine oxidase inhibition activity was demonstrated to be similar to that of the natural dipeptide, and its superoxide radical scavenging activity was enhanced above that of the natural dipeptide, suggesting that acylation did not affect the antioxidant properties of the peptide