Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions (original) (raw)
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Purification and characterisation of the water forming NADH-oxidase from Lactococcus lactis
International Dairy Journal, 2001
The NADH-oxidase (NOX) from Lactococcus lactis was isolated from extracts of aerobically grown cells. It was puri"ed by (NH ) SO fractionation followed by ion exchange and a$nity chromatographic steps. Spectra of the oxidized enzyme showed peaks with maxima at wavelengths of 274, 381 and 446 nm and FAD was identi"ed as cofactor. The enzyme is apparently a dimer composed of two identical subunits of 53 kDa, each containing one molecule of non-covalently bound FAD. NOX showed optimum activity between pH 6.0 and 9.0 and was found to have a pI of 4.8. The lactococcal NOX uses dioxygen as the natural electron acceptor and forms water during the oxidation of NADH. The natural electron donor was -NADH (K "4.1 M and V "83.2 mol min\ mg\) whereas -NADH, -NADPH or -NADPH were not oxidized by the enzyme. Hydrogen peroxide, sulfhydryl reagents and quinine inactivated the enzyme activity. The oxidase described in this paper appears to possess kinetic characteristics potentially useful to redirect, in a food-grade manner, the carbon #ux of lactococci.
Fems Microbiology Letters, 1999
The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADHoxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via K-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains. ß Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 0 9 7 ( 9 9 ) 0 0 4 3 2 -2 * Corresponding author. Instituto del Frio, Ciudad Universitaria s/n,
Fems Microbiology Letters, 2006
Product formation from glucose and NADH-oxidase activity from Lactococcus lactis MG1363, a non-citrate fermenter, was studied under aerobic conditions. In continuous cultures constantly aerated to 80% air saturation, the usual homolactic fermentation was almost completely replaced by acetate fermentation (80–90% of the fermented glucose) at low dilution rates. As the dilution rate was increased, the homolactic fermentation was partially restored, however the glucose fermentation profile was pH dependent. Significant production of acetoin and diacetyl was found at low pH and high dilution rates. NADH-oxidase activity was studied under different culture conditions. An up to fivefold increase in NADH-oxidase activity was observed in batch culture when air tension was increased from 0% to 80%. In aerobic continuous cultures at 80% air saturation, the NADH-oxidase activity was directly dependent on dilution rate and growth pH. The observed variations in NADH-oxidase activity played a crucial role in pyruvate flux redistribution and clearly correlated with the observed shifts in end product formation from glucose.
Hydrogen Peroxide Production by Lactobacillus delbrueckii Subsp. Lactis I at 5°C
Journal of Food Science, 2006
Cells of Lactobacillus delbrueckii subsp. lactis I produced hydrogen peroxide at 5°C in sodium phosphate buffer (0.2M, pH 6.5) with or without glucose. However, if the cells were starved by preincubation in buffer alone, glucose or sodium lactate were necessary to cause hydrogen peroxide production at 5°C. Hydrogen peroxide production by nonstarved cells was confirmed to be in part due to a NADH oxidase. The production of hydrogen peroxide by starved cells in buffer plus glucose in the early stage of incubation was associated with the production of a small portion of lactic acid which disappeared upon further incubation. Additional experiments revealed that hydrogen peroxide was produced in buffer containing sodium lactate added in place of glucose. Results suggested the presence of a lactate oxidase in the organism which used D-lactate to produce hydrogen peroxide.
Advanced Synthesis & Catalysis, 2005
We have successfully applied the sequence comparison-based approach to develop a peroxidase (gene AhpC) and a water-forming NADH oxidase from Lactococcus lactis (L. lactis). We found a considerably lower maximum specific activity of nox-1 (AhpF) (15 U/mg) from L. lactis compared to its nox-2 counterpart (95 U/mg). Both nox-1 and nox-2 are turnover-limited, as expected for enzymes with labile, redox-active thiols in the active site. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2: the total turnover number TTN is 82,000 for nox-1 and nox-1/peroxidase vs. 39,000 for nox-2. Addition of exogenous thiols, however, increases nox-2 stability by a factor of two, up to the level of nox-1. Kinetic and stability analysis does not reveal any clear advantage for oxygen scavenging via the nox-1 or the nox-2 routes in lactic acid bacteria. Expression levels in lactic acid bacteria upon exposure to oxidative stress rather than kinetic performance more likely account for the previously observed superiority of nox-2 effectiveness over nox-1.
Applied and Environmental Microbiology, 2002
Three isogenic strains of Lactococcus lactis with different levels of H 2 O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX ؊ strain harboring a deletion of the gene coding for H 2 O-forming NADH oxidase, and a NOX ؉ strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using 13 C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P 2 ] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P 2 depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX ؊ strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX ؉ strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX ؊ and parent strains; in the NOX ؉ strain the carbon flux was diverted to the production of ␣-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.
Oxygen Relieves the CO2 and Acetate Dependency of Lactobacillus johnsonii NCC 533
PLoS ONE, 2013
Oxygen relieves the CO 2 and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO 2 during growth. Both on agar-and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO 2 supplementation of the gas phase. Switching off the CO 2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO 2 dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO 2 requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO 2 and acetyl-CoA producing systems. Therefore, C1-and C2-compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO 2 dependency.
AFRICAN JOURNAL OF BIOTECHNOLOGY, 2011
Natural, solid and porous support named pouzzolane was used for two Lactococcus lactis subsp. lactis immobilization and lactic acid and hydrogen peroxide (H2O2) production by stains immobilization in batch fermentation process was investigated using a bioreactor with recycling. Lactic acid fermentation using syrup of molasses treated and diluted at 50% (MSTD 50%) was not supplemented by ingredients concentrations. Cells immobilization showed higher sugar utilization, higher H 2 O 2 accumulation and higher lactic acid production than did free cells. Improvement in compounds production was noticed with immobilization of Lactococcus lactis isolated from the molasses (Lc.l ML) compared with strains isolated from the cow's milk (Lc.l CM). In pure culture, the kinetic of the production of lactic acid and H 2 O 2 by the two strains in MSTD 50% was compared with than did in Elliker broth.