RESUMEN
Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.
Asunto(s)
N-Acetil Muramoil-L-Alanina Amidasa/aislamiento & purificación , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Pediococcus/enzimología , Secuencia de Aminoácidos , Cromatografía en Gel , Clonación Molecular , Estabilidad de Enzimas , Expresión Génica , Concentración de Iones de Hidrógeno , Viabilidad Microbiana/efectos de los fármacos , Micrococcus/efectos de los fármacos , Datos de Secuencia Molecular , Peso Molecular , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , TemperaturaRESUMEN
The genus Enterococcus occurs as native microbiota of fermented products due to its broad environmental distribution and its resistance to salt concentrations. Enterococcus faecalis F, a non-pathogenic strain isolated from a ripened cheese, has demonstrated useful enzymatic capabilities, a probiotic behavior and antibacterial activity against some food-borne pathogens, mainly due to peptidoglycan hydrolase activity. Its use as a natural pathogen-control agent could be further enhanced through the production of a bacteriocin, e.g. Enterocin A, because of its remarkable antilisterial activity. In this work, a markerless allelic insertion method was used to obtain an enterococcal strain capable of producing a functional enterocin. Agar diffusion tests showed that the recombinant strain was active against Staphylococcus aureus, Listeria monocytogenes and the pathogenic strain E. faecalis V583. When grown in liquid culture together with L. monocytogenes, it attained a two-log reduction of the pathogen counts in lesser time relative to the native strain. Because the DNA construction is integrated into the chromosome, the improved strain avoids the use of antibiotics as selective pressure; besides, it does not require an inductor because of the inclusion of a constitutive promoter in the construction. Its technological and antibacterial capabilities make the improved E. faecalis strain a potential culture for use in the food industry.
Asunto(s)
Bacteriocinas/genética , Bacteriocinas/metabolismo , Enterococcus faecalis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas Bacteriológicas , Cromosomas Bacterianos/genética , Técnicas de Cocultivo , Enterococcus faecalis/genética , Microbiología de Alimentos , Listeria monocytogenes/crecimiento & desarrollo , Mutagénesis Insercional , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.