A standardized assay medium to measure enzyme activities of lactococcus lactis while mimicking intracellular conditions.

A. Goel, F. Santos, W.M. Vos, B. Teusink, D. Molenaar

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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
Original languageEnglish
Pages (from-to)134-143
JournalApplied and Environmental Microbiology
Volume78
Issue number8
DOIs
Publication statusPublished - 2012

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Lactococcus lactis
enzyme activity
assay
assays
Enzymes
enzyme
Lactococcus lactis subsp. cremoris
chemostat
Ammonium Sulfate
Enzyme Assays
ammonium sulfate
enzymes
reproducibility
Freezing
freezing
glucose
cells
kinetics
Glucose

Cite this

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title = "A standardized assay medium to measure enzyme activities of lactococcus lactis while mimicking intracellular conditions.",
abstract = "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",
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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 JournalArticleAcademicpeer-review

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AU - Santos, F.

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AU - Molenaar, D.

PY - 2012

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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

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