In this paper, we report for the first time on the identification, purification, and characterization of the α-ketoisovalerate decarboxylase from
Lactococcus lactis, a novel enzyme responsible for ...the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. The
kivd gene consisted of a 1647 bp open reading frame encoding a putative peptide of 61 kDa. Analysis of the deduced amino acid sequence indicated that the enzyme is a non-oxidative thiamin diphosphate (ThDP)-dependent α-keto acid decarboxylase included in the pyruvate decarboxylase group of enzymes. The active enzyme is a homo-tetramer that showed optimum activity at 45 °C and at pH 6.5 and exhibited an inhibition pattern typical for metal-dependant enzymes. In addition to Mg
2+, activity was observed in presence of other divalent cations such as Ca
2+, Co
2+ and Mn
2+. The enzyme showed the highest specific activity (80.7 U
mg
−1) for α-ketoisovalerate, an intermediate metabolite in valine and leucine biosynthesis. On the other side, decarboxylation of indole-3-pyruvate and pyruvate only could be detected by a 100-fold increase in the enzyme concentration present in the reaction.
Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the ...2-substituted (1,3)-β-D-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages. PUBLICATION ABSTRACT
Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these β-glucans have immunomodulating properties. We show that the overproduction of the ...Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter...) of a position 2-substituted (1...3)-β-D-glucan that has potential use as a food additive. (ProQuest: ... denotes formulae/symbols omitted.)
Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the ...biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and incrementctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the incrementctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.
Lactobacillus casei subsp.
casei IFPL731 is a wild strain isolated from artisanal goats’ milk cheese. The strain shows a multiple enzymatic system that involves esterase, cell-envelope proteinase, ...aminopeptidases, dipeptidases, specialized peptidases for proline-containing peptides, and amino acid converting enzymes. The broad enzymatic system of
Lb. casei IFPL731 is responsible for its hydrolyzing activity towards a number of peptides, including bitter and methionine-containing peptides. Both characteristics are of great interest as regards the use of the strain as a starter culture adjunct to influence the development of cheese flavour, which has been demonstrated in the manufacture of goats’ milk and low fat cheeses. Moreover,
Lb. casei IFPL731 shows methionine aminotransferase activity that leads to the production of the typical cheese aroma. The different enzymes from
Lb. casei IFPL731 described in this review and its utilization as a starter culture adjunct in cheese manufacture make the strain one of the best characterized lactobacilli in the literature.
The
gfp gene from
Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in
Lactococcus lactis subsp.
lactis biovar
cremoris MG1363, upon construction and introduction of ...plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in
L. lactis.
The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of ...plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish in mixed cultures lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.
Abstract
In this paper, we report for the first time on the identification, purification, and characterization of the α-ketoisovalerate decarboxylase from Lactococcus lactis, a novel enzyme ...responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. The kivd gene consisted of a 1647 bp open reading frame encoding a putative peptide of 61 kDa. Analysis of the deduced amino acid sequence indicated that the enzyme is a non-oxidative thiamin diphosphate (ThDP)-dependent α-keto acid decarboxylase included in the pyruvate decarboxylase group of enzymes. The active enzyme is a homo-tetramer that showed optimum activity at 45 °C and at pH 6.5 and exhibited an inhibition pattern typical for metal-dependant enzymes. In addition to Mg2+, activity was observed in presence of other divalent cations such as Ca2+, Co2+ and Mn2+. The enzyme showed the highest specific activity (80.7 U mg−1) for α-ketoisovalerate, an intermediate metabolite in valine and leucine biosynthesis. On the other side, decarboxylation of indole-3-pyruvate and pyruvate only could be detected by a 100-fold increase in the enzyme concentration present in the reaction.
We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea ...victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible PM promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the PM promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the PM-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.
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