Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

Abstract

ABSTRACTKnowledge of how the activity of enzymes is affected underin vivoconditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters forLactococcus lactisare 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 ofL. lactissubsp.cremorisMG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells ofL. 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, mostin vivo-like activities were lower than previously published data. Yet, the ratios ofVmaxover measuredin vivofluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements forL. lactis.