RESUMO
Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H2O2. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H2O2 exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.
Assuntos
Catalase/genética , Catalase/metabolismo , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/metabolismo , Viabilidade Microbiana , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Catalase/biossíntese , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Lacticaseibacillus casei/efeitos dos fármacos , Lacticaseibacillus casei/enzimologia , Viabilidade Microbiana/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Superóxido Dismutase/biossíntese , Transformação BacterianaRESUMO
We previously reported that the ß-1,4-Mannanase (manB) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei. Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SPUsp45) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SPSlpA), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SPUsp45 is functionally recognized and processed by the L. casei secretion machinery. The SPUsp45-mediated secretion efficiency was â¼87%, and SPSlpA drove the export of secreted ManB with â¼80% efficiency. SPSlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.
Assuntos
Proteínas de Bactérias/genética , Vetores Genéticos , Lacticaseibacillus casei/genética , Manose-6-Fosfato Isomerase/genética , Complexos Multienzimáticos/genética , Nucleotidiltransferases/genética , Transporte Proteico , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Genes Reporter , Lacticaseibacillus casei/metabolismo , Lactococcus lactis/genética , Glicoproteínas de Membrana/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, ß-1,4-mannanase (manB) from Bacillus pumilus and ß-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Reporter , Glucuronidase/genética , Lacticaseibacillus casei/genética , Manosidases/genética , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/metabolismo , Vetores Genéticos/genética , Glucuronidase/metabolismo , Lacticaseibacillus casei/metabolismo , Manosidases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação GenéticaRESUMO
OBJECTIVE: Lactobacillus casei is widely used in food production and feed industry. The aim of this study was to construct the recombinant expression mannanase Lb. casei. METHODS: The mature peptide gene of ß-1,4-mannanase from Bacillus pumilus was cloned into expression vectors pELX1 and pELSH, then electroporated into Lb. casei, establishing an intracellular and a secretion expression mannanase Lb. casei respectively. RESULTS: After incubation, the specific activity of ß-1,4-mannanase was 23 U/mg whole cell protein for intracellular expression and 8.8 U/mL for secretion expression in supernatant. CONCLUSION: Mannanase gene expression in Lb. casei provides application prospect and deserves further study.
