Production and characterization of a novel aminopeptidase A from Lactococcus lactis / 生物工程学报

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co2+, Mn2+, and Zn2+ but was strongly inhibited by Ni2+and Cu2+. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed

Mutante de Streptococcus lactis con resistencia a bacteriófagos aislados en Argentina y Estados Unidos de América / A mutant of Streptococcus lactis with resistance to bacteriophages isolated in Argentina and the United States of America

Streptococcuslactis y S. cremoris, bacterias lácticas utilizadas para la producción de quesos, pueden reesultar infectadas por bacteriófago específicos, lo que ocasiona alteraciones en la calidad, disminución de la productividad económicas significativas. Los métodos de desinfección y control más usuales no siempre resultan en una completa protección contra la proliferación de los fagos, por los que se ha recurrido al empleo de cepas resistentes a los mismos. En el presente trabajo se ha empleado un método simple y directo para obtener mutantes espontáneas de S. lactis con resistencia a fagos. La mutante S.lacts C26 obtenida a partir de la cepa S. lactis C2 sensible a fagos exhibe propiedades bioquímicas y microbiológicas similares a las de la cepa original C2, diferenciándose de ella por una clara resistencia al fago St15, de morfología alargada, utilizado para la selección. Las curvas de la cinética de crecimiento de la mutante en ausencia y en presencia del fago resultaron semejantes. La cepa C26 fue controlada con otros dos fagos aislados en Argentina y con seis fagos de dos tipos morfológicos, aislados en la Universidad de Cornell en EE.UU. La cepa C26 que fuera aislada como mutante resistente al fafo St15, resultó también resistente a todos ellos. Por otra parte, ha sido de interés verficar que los fagos no siempre observan una especificidad estricta con respecto a la especie de la célula huésped. El fago D59-1, aislado utilizando una cepa S. cremoris, también provoca una lisis significativa en la cepa S. lactis C2, suiriendo que ambas poseen sitios receptores semejantes. La mutante C26 fue empleada con éxito en Buenos Aires, en la planta elaborada de quesos donde fuera aislado el fago St15, lo que alienta la posibilidad de emplear esta metodología en el desarrollo de fermentos lácticos