RESUMO
A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Em(r)), was constructed by ligation of pJY33 with pBluescript II SK(-) and an erythromycin resistance gene (Em(r)). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose.
Assuntos
Vetores Genéticos , Plasmídeos/genética , Weissella/genética , Composição de Bases , Replicação do DNA , DNA Bacteriano/genética , Eritromicina/farmacologia , Escherichia coli/genética , Lactobacillus plantarum/genética , Lactococcus lactis/genética , Leuconostoc/genética , Fases de Leitura Aberta , Análise de Sequência de DNARESUMO
Three Pediococcus pentosaceus strains were isolated from jeotgals, salted and fermented Korean sea-foods, and their probiotic potentials were examined. After 2 h exposure to pH 3.0, P. pentosaceus F66 survived with the survival ratio of 32.6% followed by P. pentosaceus D56 (17.2%) and P. pentosaceus A24 (7.5%). P. pentosaceus F66 also survived better (26.6%) than P. pentosaceus A24 (13.7%) and P. pentosaceus D56 (5.8%) after 2 h exposure to 0.3% bile salts. Three strains grew slowly on MRS broth with 15% NaCl (w/v), reaching the OD600 values of 0.4-0.8 in 36 h. They adhered to Caco-2 cells (10.9-13.9 CFU/cell) with similar degree of adherence of a positive control, Lactobacillus rhamnosus GG (12.8 ± 0.5 CFU/cell). Three strains possess some desirable enzyme activities such as ß-galactosidase, α-glucosidase, ß-glucosidase, and N-acetyl-ß-glucosidase. From these results, P. pentosaceus F66 seems qualified as a probiotic and can be utilized for fermented foods including jeotgals.
Assuntos
Pediococcus/fisiologia , Probióticos/farmacologia , Alimentos Marinhos/microbiologia , Aderência Bacteriana , Ácidos e Sais Biliares/metabolismo , Células CACO-2 , Enzimas/análise , Células Epiteliais/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Lacticaseibacillus rhamnosus , Viabilidade Microbiana/efeitos dos fármacos , Pediococcus/efeitos dos fármacos , Pediococcus/isolamento & purificação , Cloreto de Sódio/metabolismoRESUMO
A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.