Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc oenos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively alpha and beta. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. oenos 9204 HDC was synthesized as a precursor proHDC pi 6 (Mr 205,000). A cleavage between Ser-81 and Ser-82 generated the alpha (Mr 25,380) and beta (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (alpha beta)6. At the optimal pH of 4.8, the HDC activity exhibited a simple Michaelis-Menten kinetic (K(m) = 0.33 mmol l-1, Vmax = 17.8 mumol CO2 min-1 mg-1), while at pH 7.6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor (Ki = 32 mmol l-1). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X 2 L isolated from wine. Starved cells after 2 h of incubation at 30°C in citrate buffer, 0.05 mmol 1 −1 pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l −1 SO 2 and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l −1 Ca 2+ and Mg 2+ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3?-5?phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.