Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 134-143 |
| Journal | Applied and Environmental Microbiology |
| Volume | 78 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - 2012 |
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A standardized assay medium to measure enzyme activities of lactococcus lactis while mimicking intracellular conditions. / Goel, A.; Santos, F.; Vos, W.M.; Teusink, B.; Molenaar, D.
In: Applied and Environmental Microbiology, Vol. 78, No. 8, 2012, p. 134-143.Research output: Contribution to Journal › Article › Academic › peer-review
TY - JOUR
T1 - A standardized assay medium to measure enzyme activities of lactococcus lactis while mimicking intracellular conditions.
AU - Goel, A.
AU - Santos, F.
AU - Vos, W.M.
AU - Teusink, B.
AU - Molenaar, D.
PY - 2012
Y1 - 2012
N2 - Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of V
AB - Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of V
U2 - 10.1128/AEM.05276-11
DO - 10.1128/AEM.05276-11
M3 - Article
VL - 78
SP - 134
EP - 143
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 8
ER -