DataEntryID 102 General information Manuscript title: Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition. PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/15781419 Journal: Metabolic Engineering Year: 2005 Authors: Jiangfeng Zhu, Kazuyuki Shimizu Affiliations: Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan. Keywords: Escherichia coli, Enzyme activity, Metabolite concentration, Metabolic flux analysis, Lactate production, Pyruvate metabolism Full text article: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBc1FFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--b95290bedfea5d32f478683a0a1885ba451fb800/Zhu_2005.pdf Project name: not specified Experiment description Organism: Escherichia coli Strain: BW25113, pflA, pta, ppc, adhE and pykF mutants Data type: enzyme/protein concentrations Data units: mmol/min mg protein Execution date: not specified Experimental details Temperature (°C): 37 pH: 7.0 Carbon source: glucose Culture mode: batch Process condition: aerobic Dilution rate (h⁻¹): — Working volume: 1.0 L Biomass concentration (g/L): Cell dry weight was determined by measuring the optical density at 600 nm. See worksheet. Medium composition: not specified General protocol information: Measurement method: , colorimetric assays; Methods description: The culture broth with a volume corresponding to about 100 mg (dry weight) of cells was centrifuged (4 °C, 10 min at 10,000×g) and washed twice with 20 mM Tris-HCl buffer (pH at 7.0). Cells were resuspended in Tris (100 mM) -HCl buffer (pH at 7.0) containing KCl (20 mM), MnSO4 (5 mM), DTT (2 mM) and EDTA (0.1 mM). Cell disruption by sonication (five cycles of 30 s with 1 min cooling periods) was followed by the removal of cell debris by centrifugation for 10 min at 10,000×g at 4 °C. The supernatant was used for all enzyme assays. The protein concentration of extracts was determined by the Lowry's method [1] with bovine serum albumin used as the standard. Enzyme assays were made based on the relationships between the enzyme activity and the consumption or production of NADH or NADPH monitored at 340 nm in a spectrophotometer. The enzyme activities of glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were measured based on the methods of Moritz et al. [2]. The enzyme activities of Pyk, lactate dehydrogenase (LDH), Ack, Ppc and alcohol dehydrogenase (ADH) were measured by Riondet et al.'s method [3]. The enzyme activity of GAPDH was measured based on the method of [4]. The enzyme activity of pyruvate-formate-lyase (Pfl) was measured based on Garrigues et al. [5]. --------------------------------------------References-------------------------------------- [1] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. J. Biol. Chem., 193 (1951), pp. 265-275. [2] B. Moritz, K. Striegel, A.A. de Graaf, H. Sahm. Eur. J. Biochem., 267 (2000), pp. 3442-3452. http://doi.org/fwnmpm [3] C. Riondet, R. Cachon, Y. Waché, G. Alcaraz, C. Diviès. J. Bacteriol., 182 (2000), pp. 620-626. http://doi.org/cf5kb7 [4] S. Even, C. Garrigues, P. Loubiere, N.D. Lindley, M. Cocaign-Bousquet. Metab. Eng., 1 (1999), pp. 198-205. http://doi.org/b23hk8 [5] C. Garrigues, P. Loubiere, N.D. Lindley, M. Cocaign-bousquet. J. Bacteriol., 179 (1997), pp. 5282-5287. http://doi.org/cscc Data file: http://kimosys.org/repository/102/download?parameter=1571; http://kimosys.org/repository/102/download?parameter=1570; http://kimosys.org/repository/102/download?parameter=1219; Alternative formats: no files uploaded Submission and curation Entered by: Administrator KiMoSys Created: 2018-07-20 13:04:40 UTC Updated: 2020-09-01 20:52:16 UTC Version: 4 Status: (reviewed) 2018-07-20 13:15:12 UTC Views: 269 Downloads: 65