Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions
Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions
复制标题
DOI：
10.1128/aem.05276-11
复制
发表时间：
2012-01-01
影响因子：
4.4
通讯作者：
Molenaar, Douwe
中科院分区：
文献类型：
--
作者：
Goel, Anisha;Santos, Filipe;Molenaar, Douwe
了解酶的活性在体内条件下是如何受到影响的，对于分析它们的调节和构建产生对细胞行为的综合理解的模型是至关重要的。目前乳酸乳球菌的动力学参数分散在不同的研究中，并在不同的测试条件下进行。此外，化验条件往往与细胞内环境中的普遍条件不同。为了建立与胞内条件相似的统一的检测条件，我们分析了乳酸乳杆菌亚种厌氧葡萄糖限制的恒化培养物的胞内组成。奶油MG 1363。在此基础上，我们设计了一种新的测定乳酸乳杆菌生长细胞酶活性的测定介质，尽可能地模拟其胞内环境。对程序进行了优化，在96孔板上进行了操作，并检查了所有酶活性测量的重复性和动态范围。通过添加偶联酶，确定了冷冻和携带硫酸铵的效果。测定了10种糖酵解酶和4种发酵酶的活性。值得注意的是，大多数体内活动都低于之前公布的数据。然而，V-max与体内测量的通量之比大于1。通过这项工作，我们开发了标准的乳酸乳杆菌酶活性测量方案，并得到了广泛验证。
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-max over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis.