Difference between revisions of "Part:BBa K2835003"

Revision as of 22:22, 16 October 2018

His-tagged laccase from Trametes versicolor

This laccase from Trametes versicolor is originally a ligning degrading enzyme, but it is in fact capable of oxidizing a wide range of aromatic compounds. In our project, it has been used to inactivate the antibiotic sulfamethoxazole, one of the most persistent antibiotics found in the Baltic Sea.

Usage and Biology

Our BioBrick encodes a laccase from the fungus Trametes versicolor (PDB: 1GYC). Laccases are a class of multi-copper oxidases that fungi use to degrade lignin and be able to grow on wood. Lignin is composed of a cluster of multiple phenolic groups, which are oxidized by laccases. With their natural affinity for phenolic-like structures, laccases have also been shown to degrade a large range of other phenolic compounds. For example, many reports show that laccases can be used to degrade pharmaceuticals - including oestrogens, painkillers and antibiotics. In our project, it has been used to inactivate the antibiotic sulfamethoxazole (SMX), one of the most persistent antibiotics found in the Baltic Sea.

BBa_K2835003 was created by modification of BBa_K500002 to remove the signal peptide sequence from the native host (first 60 bp) as annotated by UniProt (accession code: Q12718). Moreover, an N-terminal 6xHis-tag were added.

In our project, we characterised this BioBrick by expressing it in the methylotrophic yeast Pichia pastoris. However, this BioBrick can be implemented in any host expression system, such as S. cerevisiae or E. coli by cloning it into an appropriate vector.

Expression

The BioBrick was cloned into the Invitrogen pPICZα A vector, which fuses it to the α-factor secretion signal from Saccharomyces cerevisiae, and puts it under control of the methanol-inducible AOX1 promoter. After confirming the cloning by sequencing, the plasmid was electroporated into the X33 Pichia pastoris strain. The transformation was confirmed by colony PCR.

Fourteen clones were picked to screen for enzyme production by cultivation in BMGY medium (containing glycerol) for 24h to gain biomass. Glycerol also derepresses the AOX1 promoter, which is repressed by glucose. Subsequently, the clones were cultivated in BMMY medium containing 1% methanol and 0.2 mM copper sulfate for 5 days. The addition of methanol induces protein production by activating the AOX1 promoter, whereas copper sulfate is required for proper folding of the laccase enzyme.

Samples were taken and analyzed daily during cultivation in BMMY by performing an ABTS activity assay (100µl 0.2mM ABTS, 800µl 100mM citrate phosphate buffer pH 4.0, 100µl supernatant). On the second day of screening, we started observing a color change in the cuvettes after 10 to 30 minutes (see Figure 1), versus no color change for the control supernatant (a blue color indicates formation of oxidized ABTS radicals). On day 4 and 5, clear increases in absorbance were observed after 30 and 60 minutes. On the fifth day the increase in absorbance was even higher (see Figure 2).

Figure 1: Cuvettes containing ABTS reactions with day 2 supernatant (labelled with clone number), after leaving to incubate at room temperature overnight. Cuvette labelled 31 contains only ABTS and buffer, cuvette labelled 32 contains only supernatant and buffer. Cuvette labelled C contains the wild type control supernatant.

Figure 2: Difference in absorbance measurements at 420nm (day 5) at t=30 min and t=60 min vs t=0 min.

Characterization

Clone 2 was cultivated on a larger scale, with daily addition of 1% methanol and copper sulfate. After 5 days, the supernatant was collected by centrifugation and snap frozen in liquid nitrogen. A control culture, which was inoculated with the untransformed Pichia pastoris X33 strain was handled in the same way.

ABTS activity assay

The biobrick was characterised by measuring laccase activity in an ABTS assay. The assay was performed in a 96-well plate, adding 240 µL citrate phosphate buffer pH 4, 30 µL 2 mM ABTS (solved in ABTS buffer) and 30 µL of the culture supernatant. Supernatant of the clone 2 culture and the control culture were measured in triplicates. The measurement was performed at 30°C.

As shown in Figure 3, the supernatant of clone 2 (expressing our BioBrick) showed a distinct activity compared to the control in the time frame of 70 min.

Figure 3: Oxidized ABTS product concentration over time in 40 µL citrate phosphate buffer pH 4, 30 µL 2mM ABTS and 30 µL culture supernatant. The assay was performed at 30°C. Triplicates were performed for both clone 2 and the control. Error bars (grey) indicate the standard deviation.

We determined K_M and v_max for our wild type laccase by performing non-linear regression of a saturation curve on experimental data (Michaelis-Menten model). The kinetic parameters found with non-linear regression were:

K_M = 0.15 ± 0.0091 mg/ml ABTS

v_max = 29 ± 0.84 μM/min

Figure 4: A comparison of the enzymatic activity of wild type laccase in different substrate concentrations at pH 4.0, 30 °C. Enzyme concentration was approximately 0.0069 mg/ml in the reactions. Error bars indicate standard deviation (n=3). The blue line represents the Michaelis-Menten model obtained from non-linear regression (R2 = 0.9952).

We were able to successfully measure the EC50 values for both the transformation reactions with our wild type laccase and pure SMX. We tested TPs generated from two different concentrations of SMX and wild type laccase; a low concentration of 5 mg/mL and a high concentration of 7.6 mg/mL SMX. Using the bioluminescent bacteria A. fischeri, we were able to measure light inhibition which correlates with A. fischeri death. SMX is toxic for A. fischeri which is shown since the EC50 values was approximately 50 mg/L for pure SMX for both 5 mg/mL and 7.6 mg/mL of SMX. For the TPs we observed a 3-fold increase of the EC50. This means that there is significant proof that the WT laccase is degrading SMX, and the products of the reaction are not toxic for the environment.

References

Sequence and Features