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General Information info

Manuscript title Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition.
PubMed ID 15781419
Journal Metabolic Engineering
Year 2005
Authors Jiangfeng Zhu, Kazuyuki Shimizu
Affiliations Department of Biochemical Engineering & 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 Downloadarticle Zhu_2005.pdf
Project name not specified

Experiment Description info

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 info

Temperature (0C) 37
pH 7.0
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 1.0
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: 2D gel-MALDI-TOF

Methods description - Notes

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

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Submission and curation info

Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-07-20 13:04:40 UTC

Updated 2018-07-20 13:14:43 UTC

Version 2

Status (reviewed) 2018-07-20 13:15:12 UTC




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