Immobilization of laccase on magnetic beads

Immobilization on Fe 3 O 4 nanosphere beads 

  

Immobilizing a laccase on Fe 3 O 4 nanosphere beads by amine coupling  

  

- Purified laccase - Fe 3 O 4 nanosphere beads - Poly(sodium 4-styrenesulfonate) PSS  - Chitosan - PBS - 1M acetic acid - Strong magnet 

    

 

1. Mix 70 mg of Fe 3 O 4 beads with 3.5mL or more of PSS solution (1wt% in deionized water) and let it shake for 1h at 200 rpm at room temperature 

2. Separate the beads with a magnet and wash the Fe 3 O 4 -PSS with deionized water 

3. Add the beads to 20 mg Chitosan in 1 M acetic acid with final volume of 50-100 mL 

4. Incubate for >18h at 200 rpm shaking 

5. Collect the beads with a magnet by holding it on the outside of the glass and discard the solution 

6. Detach the magnet and wash the coated beads with 1 mL PBS.  Attach the magnet and discard the PBS. Repeat 3-8 times 

7. Dry the beads at 40°C for 8-10 hours 

8. Prepare Glutaraldehyde (GA) solution with 5 mL of 50% wt in 5 mL PBS   

9. Mix 0,5 mL of 25% wt GA solution in 10 mL PBS (pH 7.0) 

10. Mix ~70 mg of the beads in 10 mL of GA solution for 1h on a shaker at room temperature and collect the GA linked chitosan-coated beads (Fe 3 O 4 -PSS-CH->GA) with a magnet  

11. Wash the beads with deionized water 3-8 times 

12. Suspend Fe 3 O 4 -PSS-CH->GA in 15 mL PBS (pH 7.0) 

Immobilization of laccase on magnetic beads

13. Add 15 mg laccase in solution to get 1mg/mL 

14. Shake solution for 15-18h overnight 

15. Collect beads with a magnet and wash the beads with 10 mL PBS. Attach the magnet and discard the liquid. Repeat 3-8 times. 

16. Store the dry beads at room temperature.  

17. For activity assay: Leave the beads with the laccase at least for 6h in the pH you want to test before you start the activity assay.  

   

The immobilization principle is based on amine coupling. Primary amines of Chitosan coupled to Glutaraldehyde and primary amines provided by Lysine residues of the protein are coupled to the aldehyde group of Glutaraldehyde.   

  

Yeon K-M, Lee C-H, Kim J. Magnetic Enzyme Carrier for Effective Biofouling Control in the      Membrane Bioreactor Based on Enzymatic Quorum Quenching. Environmental Science      & Technology. 2009;43(19):7403–9.  Yufang Zhu, Stefan Kaskel, Jianlin Shi, Tobias Wage, and Karl-Heinz van Pée Immobilization      of Trametes versicolor Laccase on Magnetically Separable Mesoporous Silica Spheres      Chemistry of Materials 2007 19 (26), 6408-6413 DOI: 10.1021/cm071265g  

 

Immobilization on Dynabeads  

 

Immobilizing a laccase on streptavidin-coated Dynabeads®.

- Purified laccase 

Immobilization of laccase on magnetic beads

- Magnetic rack - Streptavidin-coated Dynabeads® from ThermoFisher - Biotin-XX Microscale protein labeling kit - PBS 

      I. Labeling reaction

1. Add 1 ml of deionized water to the vial of sodium bicarbonate (component B). Vortex or pipette up and down until dissolved.

2. Transfer the dissolved protein to the reaction tube (component A). Add 1/10 of the protein volume of component B and mix by pipetting up and down.

3. Add 10 µl deionized water to the vial of Biotin-XX, SSE (component A/Reactive biotin). Dissolve completely by pipetting up and down. Discard the leftover biotin.

4. Add calculated volume of reactive biotin to protein and mix thoroughly by pipetting up and down several times.

5. Incubate at RT for 15 min. 

 II. Purification

1. Resuspend the gel resin (component E) by gently rocking it. Fill the upper chamber of a spin filter (component D) to the upper lip with gel resin (ca 800 µl). Centrifuge at 16000 x for 15 seconds. Prepare one for every 50 µl of sample. (or reuse the same)

2. Rinse out the collection tube several times if resin is present and replace the resin containing insert if needed.

3. Pipette 50 µl of labeled protein mixture onto each resin and centrifuge at 16000 xg for 1 min. (or reuse)

4. Each tube now contains purified biotinylated protein.

  III. Immobilization

1. Add 150 µl Dynabeads® M-280 Streptavidin to an Eppendorf tube. Add 850 µl PBS, mix, and place the tube in a magnetic rack.

Immobilization of laccase on magnetic beads

2. When the beads have gathered at the magnet, remove the supernatant and resuspend the beads in 150 µl PBS.

3. Add 25 µl of washed beads to a tube and remove the supernatant. Add 1 µg of biotinylated protein, and add up to a total volume of 25 µl with PBS.

4. To investigate laccase activity add 900µL of and 100µL of ABTS. Wait for color change and place on magnetic rack for separation.

If the decreasing amount of protein available is to be demonstrated:

1. Add 25 µl of washed beads to a new tube and remove the supernatant. Add 1 µg of biotinylated protein, and add up to a total volume of 25 µl with PBS. Incubate on rotamixer at RT for 30 min.

2. Place the tube and a new tube containing 25 µl washed beads in the magnetic rack. When the beads have gathered at the magnet, discard the supernatant in the new tube and transfer the first supernatant to the new tube. Incubate the new tube on rotamixer at RT for 30 min. Resuspend the beads from the step 3 in 25 µl PBS.

3. Repeat once more with 25 µl and after that with the 75 µl that is left. The last supernatant should be transferred to a new Eppendorf tube without beads.

4. Add 6 µl 4X SDS RED loading buffer to all tubes and denature the samples at 95 °C for 5 min. Place the tubes on magnetic rack and load samples without beads on an SDS-gel. Run the gel at 220V for 20-35 min and stain.

Biotin also binds to primary amines provided by Lysines on the outer part of the protein.

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