Team:Uppsala Universitet/Demonstrate


HRP extracted from horseradish

Due to bottlenecks in enzyme supply, HRP was isolated from locally purchased horseradish. Crude extract was prepared by blending, sonication and subsequent ammonium sulphate precipitation (30 % and 65 % saturation). 65 % ammonium sulfate fraction was resuspended in 50 mM phosphate buffer pH 7,5. Enzyme activity was confirmed by ABTS-assay, which led to an immediate change in the color (Figure 1B). When the assay was tested on the extracted HRP, a dilution of the sample was made to 0.0005 before applied to the cuvette and tested with spectrophotometer (Figure 1B). The HRP oxidizes the ABTS which leads to a colorimetric change of the sample.
The enzymes AAO and HRP was expressed from Pichia Pastoris. The absorbance measured can be observed in the graph below (Figure 3).

Figure 1: Activity of horseradish peroxidase extracted from horseradish.
(A) Color-change after addition of undiluted crude Horseradish extract into ABTS reaction mixture. (B) The increase in absorbance indicates HRP activity.

Figure 2: ABTS-assay of bought HRP
The figure shows ABTS-based assay on commercial HRP with absorbance plotted (405 nm) against time (s). A clear increase in absorbance after addition of enzyme indicates that activity is detectable.

Commercial HRP

After commercial HRP was obtained, an ABTS-based assay was performed to assess the activity (Figure 2). A dilution of 1:200 for the bought HRP was made before applying the sample and working reagent into a cuvette and analyzed in a spectrophotometer. The result indicates that the assay is able to detect the presence of HRP, which in turn means that the activity of yeast-expressed HRP would be detectable.
The enzymes AAO and HRP was expressed from Pichia Pastoris. The absorbance measured can be observed in the graph below (Figure 3).

Figure 3: Assays of cell lysate for HRP and AAO enzymatic activities
The activities of the two enzymes expressed in Figure 3 were assessed using two different enzymatic assays. Uninduced, orange; induced, green. Assays were performed in triplicates. (A) ABTS-assay for HRP. (B) FOX-assay for AAO activity. Two different volumes of lysate were used and absorbance at 560 nm was measured. The difference in activity between uninduced and induced samples was statistically significant as measured by paired t-test (*= p<0.05 and ****= p<0.00001, respectively).

Treatment of Kraft Lignin with HRP

With the goal to enzymatically degrade lignin, bought HRP and hydrogen peroxide was added to lignin in water. A change in color was observed in the samples over time, which could be indicative of a change in the lignin structure (Figure 4).

Figure 4: Three samples of Kraft Lignin treated with bought HRP.
The samples have been exposed to HRP for different amounts of time. A change in color can be observed in the samples that have been exposed to HRP for a longer time.