Regulation of alpha-ketoisovalerate decarboxylase expression in Lactococcus lactis IFPL730.

The alpha-ketoisovalerate decarboxylase (Kivd) is a unique lactococcal key enzyme in the decarboxylation of branched-chain alpha-keto acids derived from branched-chain amino acids transamination into aldehydes. These products are important aroma compounds produced during cheese ripening. In this study, the Kivd expression of Lactococcus lactis IFPL730 growing either in nitrogen-rich media or in chemically defined media supplemented with different concentrations of branched-chain amino acids and casitone was analyzed. Isoleucine starvation increased 4-fold the Kivd activity of L. lactis IFPL730 compared to cells grown in chemically defined medium with casitone as nitrogen source. Regulation of expression was at the transcription level, probably mediated by CodY, for which a consensus CodY-box sequence was identified in the kivd promoter region.

Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis.

In this paper, we report for the first time on the identification, purification, and characterization of the alpha-ketoisovalerate decarboxylase from Lactococcus lactis, a novel enzyme responsible for the decarboxylation into aldehydes of alpha-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 alpha-keto acid decarboxylase included in the pyruvate decarboxylase group of enzymes. The active enzyme is a homo-tetramer that showed optimum activity at 45 degrees 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 Umg(-1)) for alpha-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.

Enhancement of 2-methylbutanal formation in cheese by using a fluorescently tagged Lacticin 3147 producing Lactococcus lactis strain.

The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains L. lactis T1 and L. lactis IFPL730, showing branched chain amino acid aminotransferase and alpha-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.