Measurement of the Galactanase Activity of the GanB Galactanase Protein from Bacillus subtilis

Materials and Reagents

1. Eppendorf tubes 1.5 ml
   
2. TLC-plate: Silica Gel 60 F₂₅₄ (Merck Millipore, Darmstadt, Germany)
   
3. Sodium acetate trihydrate (Carl Roth, catalog number: 6779 )
   
4. Galactan from lupin (Arabinofuranosidase treated lupin pectic galactan, Gal:Ara:Rha:Xyl: GalUA = 91:2:1.8:0.2:5.0) (Megazyme, catalog number: P-GALLU )
   
5. β-1,4-galactobiose (Sigma-Aldrich, catalog number: G9662 )
   
6. D-(+)-galactose (Sigma-Aldrich, catalog number: G5388 )
   
7. 3,5-dinitrosalicylic acid (Sigma-Aldrich, catalog number: D0550 )
   
8. Potassium-sodium-tartrate tetrahydrate (Carl Roth, catalog number: 8087 )
   
9. Sodium sulfite (Carl Roth, catalog number: P033 )
   
10. Sodium hydroxide 50% (Carl Roth, catalog number: 8655 )
    
11. AZCL-galactan: Azurine-crosslinked-potato (AZCL) galactan (which has been purified from potato fibre and treated to remove most of the arabino- furanosyl residues) (Sigma-Aldrich, catalog number: 38127 )
    Note: This product has been discontinued.
    
12. Ammonium heptamolybdate tetrahydrate (Carl Roth, catalog number: 7311 )
    
13. Cerium sulfate tetrahydrate (Carl Roth, catalog number: P014 )
    
14. Sulfuric acid (H₂SO₄)
    
15. Potassium-phosphate dibasic (KH₂PO₄) (Carl Roth, catalog number: P018 )
    
16. di-Potassium hydrogen phosphate (K₂HPO₄) (Carl Roth, catalog number: P749 )
    Note: Prepare a 0.1 M solution in pure H₂O and use it for preparation of the potassium phosphate buffer described in Recipes section.
    
17. Sodium borate decahydrate (Sigma-Aldrich, catalog number: G5388 )
    
18. Sulfuric acid 95% (Carl Roth, catalog number: HN62 )
    
19. Acetone (Carl Roth, catalog number: 9372 )
    
20. Milli-Q pure H₂O
    
21. Purified recombinant His₆-tagged GanB (see Recipes)
    Note: This protein was produced in E. coli JM109 harboring the plasmid pHWG1119 after induction with rhamnose as described by (Watzlawick et al., 2016). Crude extracts were purified by immobilized-metal affinity chromatography using TALON^® Metal Affinity resins (Clontech Laboratories, USA) according to the supplier’s instruction. Fractions of the purified proteins were combined and the imidazole was removed with NAP10 columns (GE Healthcare, Munich, Germany) equilibrated with 0.1 M KPP.
    
22. Lupin-galactan (see Recipes)
    
23. DNS-reagent (see Recipes)
    
24. AZCL-galactan (see Recipes)
    
25. TLC developing reagent (see Recipes)
    
26. Potassium phosphate buffer (KPP), 0.1 M (see Recipes)
    
27. 0.4 M sodium borate solution (see Recipes)
    
28. 0.3 M sodium acetate, pH 5.2 (see Recipes)
    
29. TLC mobile phase solution (see Recipes)
    
30. HPAEC isocratic mobile phase (see Recipes)

Equipment

1. Pipette P2 , P20 , P200 , P1000 (Gilson Pipetman classic)
   
2. Vortex Minishaker (Heidolph Instrument, model: Heidolph Reax 2000 )
   
3. Centrifuge (Eppendorf, model: 5415 D )
   
4. Thermomixer compact (Eppendorf)
   
5. Blow-dryer
   
6. HPLC apparatus: The HPLC apparatus consists of a pump (220, Bischoff, Leonberg, Germany) and an ESA Coulochem II electrochemical detector (Bischoff, Leonberg, Germany) with an analytical cell (5040, ESA Coulochem II, Bischoff)
   
7. HPLC-column (Thermo Fisher Scientific, model: Dionex^TM CarboPac^TM PA1 )
   
8. TLC-chamber (size: 12 x 12 x 7 cm) (Desaga, Heidelberg, Germany)
   
9. Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: GENESYSTM 10 Vis )
   
10. pH meter (Metteler Toledo, model: FE20 ATC FiveEasy^TM )
    

Procedure