Enzymatic synthesis of new antimicrobial peptides for food purposes

Abstract

Growing consumer awareness of the potential negative health effects of synthetic antibiotics has prompted the search for more natural preservatives that can improve the safety and quality of food. In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Ile-Gln (Z-IQ) which is the precursor of Ile-Gln (IQ), a new antibacterial dipeptide, using an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate in 0.1 M Tris - HCl buffer pH 8. A partially purified proteolytic extract from the fruits of Solanum granuloso leprosum, named granulosain, proved to be a robust biocatalyst for the synthesis of Z-IQ, eliciting 71 ± 0.10% maximal peptide yield in the above described conditions. After cleaving and purifying IQ dipeptide, antimicrobial activity was assayed against Staphylococcus aureus ATCC 25923, Staphylococcus hominis A17771, and Staphylococcus aureus C00195, and MIC values between 118 ± 0.01 μg/mL and 133.7 ± 0.05 μg/mL were obtained. In addition, IQ showed MIC of 82.4 ± 0.01 μg/mL and 85.0 ± 0.00 μg/mL against Escherichia coli ATCC 25922 and Escherichia coli A17683, respectively. IQ did not show inhibitory activity against single-drug resistance (SDR) strains, such as Klebsiella oxytoca A19438 (SDR) and Pseudomonas aeruginosa C00213 (SDR), and against multidrug-resistant Enterococcus faecalis I00125 (MDR). IQ also caused growth inhibition of Helicobacter pylori NCTC 11638 and three wild-type H. pylori strains, which are sensitive to AML, MTZ, LEV and CLA (H. pylori 659), resistant to LEV (H. pylori 661 SDR), and resistant to MTZ (H. pylori 662 SDR). Finally, this study contributes with a new dipeptide (IQ) that can be used as an antimicrobial agent for food preservation or as a safe ingredient of functional foods.

Keywords: Ile-Gln (IQ); antibacterial peptide; enzymatic synthesis of peptides; food preservation; novel peptide against sensitive and SDR Gram positive and Gram negative strains.

Figure 1

Preferences of granulosain for N-α-[Carbobenzyloxy]-amino acid-p-nitrophenyl esters (Z-AA-ONp) in 50% (v/v) ethyl acetate in 0.1 M Tris–HCl buffer pH 8, at 40°C.

Figure 2

Component separation by RP-HPLC of a representative sample from N-α-CBZ-Ile-Gln-OH (Z-IQ) enzymatic synthesis under kinetic control, using granulosain (2.47 mg/mL, 32.49 ± 0.186 IU/mg) as soluble biocatalysts in 50% (v/v) ethyl acetate in 0.1 M Tris–HCl buffer pH 8, after 1 h of reaction at 40°C and 200 rpm. I: granulosain (t_R: 2,7–3,2 min); II: N-α-CBZ-Ile-OH (Z-I-OH) (t_R: 4 min); III: N-α-CBZ-Ile-Gln-OH (Z-IQ) (t_R: 6.5 min); IV: 4-Nitrophenol (ONp) (t_R: 8.0 min); V: N-α-CBZ-Ile-ONp (Z-I-ONp) (t_R: 15.2 min). Aqueous phase: pink line. Organic phase: black line.

Figure 3

Chromatogram obtained by (A) RP-HPLC and (B) mass spectrum of the carboxyl terminal peptide N-α-CBZ-Ile-Gln-OH (Z-IQ). The mass spectrum of the main product of the enzymatic synthesis reaction (III, t_R: 6.5 min) showed ion mass (m/z): 393, corresponding to the peptide Z-IQ.

Figure 4

Mass spectrum of the amine-terminal IQ peptide, m/z: 259.3.

Figure 5

Growth kinetics in batch culture of (A) Staphylococcus aureus C00195, using Müller-Hinton broth and increasing concentration of IQ (0 to 118 μg/mL) at 37°C and 180 rpm. (B) Helicobacter pylori NCTC 11638, using Müller-Hinton broth supplemented with 5% fetal bovine serum, and increasing concentration of IQ (0 to 250 μg/mL), at 37°C under microaerophilic conditions. Control (without IQ).

Figure 6

Growth kinetics in batch culture of Escherichia coli ATCC 25922, using Müller-Hinton broth and increasing concentration of (A) IQ (0 to 164.8 μg/mL). (B) Z- IQ (0 to 164.8 μg/mL), at 37°C and 180 rpm. Control (without IQ).