Summary

[http://www.sigmaaldrich.com/catalog/product/sigma/51639?lang=de&region=DE TVEL0] was characterized in terms of its activity to establish activity test protocols and to create a standard which can be used as a reference.

TVEL0 Activity Tests

Initial Activity Test

Figure 1:Activity of TVEL0 laccase using 0.1 mM ABTS in 100 mM sodium actetate buffer (pH 5) measured at 25°C over a time period of 5 minutes. Saturation takes place after 3 minutes with 80% ABTS oxidized.

To standardize activity test methods used for this project a [http://www.sigmaaldrich.com/catalog/product/sigma/51639?lang=de&region=DE laccase from Trametes versicolor] (TVEL0) was used. The optimal composition for activity measurements contains 140 µL of 0.03 mg mL^-1 concentrated TVEL0 laccase solution, 100 mM sodium acetate buffer, 0.1 mM ABTS, ad 200 µL H₂O. With this approach activity in oxidizing ABTS of TVEL0 was measured over a time period of 5 minutes at 25°C. The saturation of the reaction was reached after 3 minutes when ~80% ABTS got oxidized (see figure 1). This result proofed the activity measurement method and can therefor be used as a positive control.

Optimal pH of TVEL0

Figure 2:Activity of TVEL0 laccase using 0.1 mM ABTS in 100 mM sodium actetate buffer testing a pH range from pH 1 to pH 9. Measurements were done at 25°C over a time period of 5 minutes. Only at pH 5 a saturation takes place after 3 minutes with 80% ABTS oxidized.

To determine the activity of TVEL0 in regard of optimal pH conditions different sodium acetate buffer pHs were under consideration for activity tests. Ranging from pH 1 to pH 9 the standardized activity setup of 100 mM sodium acetate, 140 µL of 0.03 mg mL^-1 concentrated TVEL0 laccase solution, 0.1 mM ABTS, ad 200 µL deionized H₂O was used. Only at pH 5 a saturation in ABTS_ox can be reached (see figure 2). As in the initial activity test above the maximal amount of ABTS_ox accounts ~80%. In summary the optimal pH for TVEL activity in oxidizing ABTS is ph 5.