Immobilization

Immobilization on silica beads

• Suspend silicium dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl2) and mix it with the 1 mg/mL lacasse solution
  • Ratio of beads to protein should be 1 to 1000
  • 0.1 mg/mL final protein concentration
  • Contact with recrystallization buffer will start assembly of SbpA
• Incubate on vertical rotator at room temperature for 4 h
• After incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH2O and store them afterwards in ddH2O at 4 °C in the dark

Immobilization with CPC silica beads

• CPC-(controlled pore carrier) Silica beads, aminopropyl derivate (2-aminopropyl-triethosysilane), pore size: 500 A, 30-45 mesh

Immobilization of 1mL protein solution on 0,12g CPC-silica beads

• Immerse beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours.
• Wash beads (2 times with 1 ml) with Britton-Robinson-buffer (pH 5).
• Immerse in stock laccase solution (i.e., 1 mg of laccase in 1 mL of buffer prepared the same day) for at least 24 hours at 4° C.
• Wash the beads with buffer again (2 times with 1 mL).
• Add 1 mL 0.5M sodium chloride.
• Wash with buffer again
• Immerse in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C.
• Wash first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use.

All reagents used in the immobilization process were made in Britton-Robinson-buffer.

Activity measurements

Activity measurement of beads

Activity measurements of laccases bound on beads were performed using [http://www.pall.com/main/Laboratory/Product.page?id=20000 AcroPrep^TM 96-well Filter Plates].

• 158 µL deionized H₂ and 40 µL 100 mM sodium acetate were added to the samples containing dry beads.
• The whole sample volume was transfered into the [http://www.pall.com/main/Laboratory/Product.page?id=20000 AcroPrep^TM 96-well Filter Plates].
• The reaction was induced adding 0.1 mM ABTS at room temperature.
• The reaction was stopped by centrifugation at 13000 g for 30s.
• Oxidized ABTS was detected photometrically at 420 nm.

Alternative measurement of beads

Activity measurements of laccases bound on beads were alternatively performed while using the photometer Thermo biomate3 UV-Vis Spectrophotometer and classic plastic cuvettes.

• Beads were measured under substrate saturation in a total volume of 500 µl.
• The reaction was induced adding the specific ABTS concentration at room temperature.
• Mixing was realized through vortexing between measurements.
• Substrate reduction was measured every 20 seconds for at least an hour.
• Oxidized ABTS was detected photometrically at 420 nm.

Activity measurement of supernatant

Activity measurements of the supernatants see [here]