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Immobilization on silica dioxide beads

Immobilization on CPC-beads

Improvement of bead concentration and incubation time

To improve the immobilization proses were different concentrations of CPC-beads incubated with 1 mL TVEL0 solution for 36 hours. The protein concentration was measured with Roti®-Nanoquant.

To determine the optimal ratio of s CPC-beads to protein for immobilization, the binding capacity (Bc) is plotted against the concentration of CPC-beads.

Bielefeld2012 Immobilisierung bindingcapacity.jpg

The binding capacity shows that the best immobilization result is reached with a CPC-bead concentration of 0.12 g/ mL. Therefor we used this concentration for further experiments.

File:Bielefeld2012 improvement-of-beat-concentration.jpg
Fig. Binding capacity of different CPC-bead concentrations plotted against the concentration of CPC-beads used to immobilize TVEL0, shown for the Improvement of the immobilization strategy while using different beat concentrations and incubation for 36 hours

To optimize the incubation time was the optimal beat concentration of 0.12 g/ mL (fig. …) incubated for different time periods in 1 mL of the same protein solution that was used for the improvement of the beat concentration. Fig. … shows the mass of bound protein [µg] per g beats.

File:Bielefeld2012 improvement-of-incubation-time.jpg
Fig. Mass of bound protein [µg] per g beats incubated for different time periods to optimize the incubation time.

As can be seen binds the main concentration of protein during the first 12 hours of incubation. Because of the used literature we incubated fist for 18 and 36 hours, and chose 24h of incubation for further experiments.

Immobilization behavior

To analyze the immobilization behavior of the CPC-beads for different lacasses were TVEL0, ECOL and BPUL immobilized on CPC-beads. Therefor we used a beat concentration of 0.12 g/mL with an incubation time of 24 hours. The protein concentration was measured with Roti®-Nanoquant. In fig. … is the protein concentration in the supernatant shown of a percentage of the inserted protein concentration.

File:Bielefeld2012 protein-concentration.jpg
Fig... Protein concentration in % of the original inserted Protein concentration in the supernatant after immobilization of TVEL0, ECOL and BPUL on 0.12 g/ml CPC-beads and incubation for 24 hours.

Fig.. show that the best immobilization result is reached with ELOL with 0.2 % of the original inserted protein concentration left in supernatant of immobilized CPC-beads. The TVEL0 shows a remaining of proteins in supernatant of 12.4 % of the original inserted protein concentration and BPUL 74,2 %.


Table … shows the real inserted protein concentrations. Because of the low protein concentration in the used lacasse solutions, were they used directly, which means that it was not possible to immobilize the same concentration of all three lacasses.

Enzyme activity of immobilized Lacasses

To analyze the activity of the bound lacasses were they immobilized on 0.12 g/ mL beats and incubated for 24 hours. The activity of the beats was measured over a period 65 minutes in case of TVEL0 and over 180 minutes in case of ECOL and BPUL.


[1]Fernández-Fernández M et al. (2012) Recent developments and applications of immobilized laccase. Biotechnol Adv. 2012 Feb 28. [Epub ahead of print]

[2]P.-P. Champagne and J.A. Ramsay (2007) Reactive blue 19 decolouration by laccase immobilized on silica beads. Appl Microbiol Biotechnol. Oct;77:819–823

[3]Chantale Cardinal-Watkins and Jim A. Nicell (2011)Enzyme-Catalyzed Oxidation of 17ß-Estradiol Using Immobilized Laccase from Trametes versicolor. Enzyme Research, vol. 2011, Article ID 725172, 11 pages