Antimicrobial peptides produced by Lactobacillus sakei 2a: cloning and heterologous production.

KATIA GIANNI DE CARVALHO; MONIKA FRANCISCA KRUGER; EMILIANO SALVUCCI; FERNANDO SESMA; BERNADETTE DORA GOMBOSSY DE MELO FRANCO

Holanda

Congreso; 2nd TNO Beneficial Microbes; 2010

Strain Lactobacillus sakei 2a, isolated from a Brazilian pork product [1], is capable of producing several antimicrobial peptides, active against Listeria monocytogenes in vitro, in situ and in vivo. Activity of Lactobacillus sakei 2a in foods as biopreservative is limited, so the use of partially purified peptides as food additives may result in better inhibition. Three peptides are constantly produced: one is the classical sakacin P, the second is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23K, and the third is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23K. Their molecular masses are 4.4, 6.8 and 9.5 kDa, respectively. Purification of these peptides is cumbersome, and cloning of the genes responsible for their production and heterologous expression in Escherichia coli may be an alternative for enhanced production of these peptides. In this study, total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of the three antimicrobial peptides. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. Supernatants of cultures of these cells obtained after their rupture were active against L. monocytogenes Scott A. The molecular masses of the cloned peptides were confirmed by mass spectrometry.