Determination of Fructokinase Activity from Zobellia galactanivorans
检测Zobellia galactanivorans的果糖激酶活性
发布: 2015年11月05日第5卷第21期 DOI: 10.21769/BioProtoc.1633 浏览次数: 9462
评审: Valentine V TrotterChristian RothAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of Zobellia galactanivorans. The ZgFK enzyme catalyzes the conversion of fructose to fructose-6-phosphate using ATP as a cofactor.
[Principle] Fructokinase (FK) activity was determined by an enzyme-coupled assay (Figure 1). ADP formed through the FK reaction, i.e., phosphorylation of fructose to fructose-6-phosphate (F6P), is used by the pyruvate kinase (PK) which transforms phosphoenolpyruvate (PEP) to pyruvate. Then, lactate dehydrogenase (LDH) converts pyruvate to lactate using NADH as a cofactor. FK activity is measured by following the decrease in absorbance at 340 nm which corresponds to the transformation of NADH to NAD+.
Figure 1. Enzyme-coupled reaction used for determination of fructokinase (FK) activity
Fructokinase (果糖激酶)
Zobellia galactanivorans (zobellia galactanivorans)
Flavobacteria (黄杆菌)
Materials and Reagents
- UV-Star® PS Microplate (96 Well) (Greiner Bio-One GmbH, catalog number: 655801 )
- Purified recombinant His-tagged ZgFK
Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ZgFK vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare Dharmacon) onto an Äkta avant system (GE Healthcare Dharmacon). The complete purification protocol is described in details in Groisillier et al. (2015). - Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle (Sigma-Aldrich, catalog number: P0294 )
- MilliQ water
- Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt (Sigma-Aldrich, catalog number: P7252 )
- Trizma® base (Sigma-Aldrich, catalog number: T1503 )
- 4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
- Bis-Tris (Sigma-Aldrich, catalog number: B9754 )
- β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
- Examples of chemicals to be tested to assess substrate specificity:
- D-(-)-fructose (Sigma-Aldrich, catalog number: F0127 )
- D- (+)-glucose (Sigma-Aldrich, catalog number: G8270 )
- D-(+)-mannose (Sigma-Aldrich, catalog number: M4625 )
- D-mannitol (Sigma-Aldrich, catalog number: M9647 )
- D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
- D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
- D-fructose-1-phosphate (Sigma-Aldrich, catalog number: S408689 )
- α-D-Glucose 1-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9380 )
- D-Mannose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: M3655 )
- D-Glucose 6-phosphate sodium salt (Sigma-Aldrich, catalog number: G7879 )
- D-Fructose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: F3627 )
- Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: A1852 )
- D-(-)-fructose (Sigma-Aldrich, catalog number: F0127 )
- 1 M Tris-HCl (pH 7.5) (see Recipes)
- 100 mM MgCl2 (see Recipes)
- 1 M KCl (see Recipes)
- 100 mM PEP (see Recipes)
- 100 mM ATP (see Recipes)
- 10 mM NADH (see Recipes)
- PK/LDH (see Recipes)
Equipment
- Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
- NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)
Software
- Hyper 32 (Hyper32.software.informer.com)
- Microsoft Excel
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Groisillier, A. and Tonon, T. (2015). Determination of Fructokinase Activity from Zobellia galactanivorans. Bio-protocol 5(21): e1633. DOI: 10.21769/BioProtoc.1633.
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分类
微生物学 > 微生物生物化学 > 糖类
生物化学 > 糖类 > 甘露醇
生物化学 > 蛋白质 > 活性
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