Team:Bielefeld-Germany/Results/immo

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Immobilization

Summary

Zusammenfassung

Contents


First Approach

The first step was to find out a convenient method of immobilization using commercially acquired laccases from Trametes versicolor ( named TVEL0) as a standard. The first alternative was the use of silica dioxide beads which were already available in the lab. Different bead concentrations were used (ratio 1:500, 1:1000 and 1:1500) and different buffers (HBSS buffer, recrystallization buffer and Britton-Robinson Buffer). However, no significant activity could be detected. Therefore, it was agreed to try out CPC-(controlled pore carrier) silica beads, to which laccases covalently bind, especially that some papers with protocols and activity tests proving the efficiency were available.

Immobilization on silica dioxide beads

Immobilization Strategy and Optimization of CPC-beads:

In order to identify the best conditions for TVE01, different bead concentrations: 0.01, 0.02, 0.04, 0.06, and 0.08 g/mL, as well as different incubation time periods: 18h and 36h were examined. Britton-Robinson buffer (pH 5) was used, since TVE01 showed the highest activity in a pH 5. The beads were first immersed in 2.5% glutaraldehyde and incubated for 2 hours under light vacuum in order to allow as much beads’ surface area to be coated with aldehyde groups, which crosslink the laccases to the beads. After that, the beads were washed 3 times with Britton-Robinson Buffer and then immersed in 1mg/mL laccases from TVEL0 and placed on a rotator at 4°C for 18h and 36h respectively. After incubation, the supernatants were gathered to be tested for laccase activity. The beads were then washed with Britton-Robinson Buffer, then with 0.5M NaCl solution in order to wash away all noncovalently bound laccases and again twice with the same buffer. Subsequently, the beads were immersed in 2.5 mg/mL glycine for 18h at 4°C. Finally, the beads were rinsed again with with Britton-Robinson Buffer, then with 0.5M NaCl solution and again twice with the same buffer.

The activity of the nonbound laccases present in the supernatant was measured using ABTS as substrate. After comparing the results, it was obvious that the activity of the supernatant after 36h was less than after 18h, which indicated that after 36h, more laccases were bound to the beads. On the other hand, the activity also decreased upon increasing the concentration of the beads without reaching a saturation level (see Fig. 1). Therefore, another experiment was carried out with higher bead-concentrations.


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.

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.

300px|left|thumb|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.

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


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.

File:Bielefeld2012 enzymatic-immobilized-TVEL0.jpg
Fig. ...Enzymatic activity of immobilized TVEL0 shown as oxidized ABTS [µM] during a time period of 65 minutes.


fig.... Oxidized ABTS [µM] over a time period of 180 minutes from on CPC-beads immobilized ELOL and BPUL.


Enzyme activity of supernatant

Enzyme activity of inserted Lacasse solutions

Conclusion

Literature

[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


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