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 biomate3 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]