Proof of Concept



PETALUTION is an ambitious project attempting to solve PET and heavy metal pollution in Ghana and beyond. Whilst not every aspect of our project was successful, we were able to get promising data for our PET biodegradation and bioremediation devices. Both these devices involve immobilising an enzyme onto a matrix and we were able to prove that we could get active enzymes on these matrices to then degrade PET and remediate heavy metals respectively.

Bioremediation

We can demonstrate that we have created hydrogels (3C hydrogels) that are able to remediate heavy metal out of contaminated water. As these hydrogels are designed to be cell-free they are also viable for in-field use.

Zinc (II) and Nickel (II) Binding Capacity of Metallothionein-Displaying 3C Hydrogels

To demonstrate that our Metallothionein-Displaying 3C Hydrogels and 3C hydrogels can chelate heavy metals we used inductively coupled plasma mass spectrometry (ICP-MS) to measure solutions of Zinc (II) and Nickel (II) as ICP-MS measures the concentration of a certain metal ion in solution. We then placed our hydrogel into this solution and then measured the metal concentration after the hydrogels have been in the solution for an hour Therefore, the reduction in the number of unbound metal ions in the supernatant represents the number of metal ions sequestered in the 3C hydrogels (Figure 1).

Figure 1. Amounts of (A) Zn (II) and (B) Ni (II) ions captured by 3C hydrogels. The 3C hydrogel fragments were washed with the buffer (0.4 M Tris-HCl, pH 7.5) 3 times, then incubated in 1 ml of 100 uM Zn (II) or Ni (II) solution (using the buffer as the solvent). The values were obtained by multiplying the molecular weight of the metal ion with the difference between the initial and final concentrations of metal ion in the supernatant, which were measured using ICP-MS. Because of varying weights of the hydrogel fragments, the quantity of metal ion sequestered was divided by the weight of the hydrogel fragment used and multiplied by 20 to calculate the data representative of a 20 mg hydrogel fragment.

Figure 1 shows that both our 3C hydrogels and Metallothionein-Displaying 3C Hydrogels can bind and chelate heavy metals. However, we saw no significant difference between the 3C hydrogel control and the 3C hydrogel decorated with CBD-tagged metallothionein. The most probable reason for this would be that the hydrogel matrix plays a major role in metal ion sequestration, as we saw this effect in earlier experiments. Another reason may be that the CBD tag is affecting the metallothionein folding and therefore reducing its metal binding capacity (See results section for more information). Despite this we still have hydrogels which can remediate heavy metals and be used as part of a heavy metal bioremediation device.

If we wanted to improve our current 3C-hydrogel we would focus on improving the metallothionein function. To do this we would use purified proteins instead of crude lysates as they are too impure and contain metalloenzymes and metal-chelating proteins which could affect our results. We could also try to optimise the expression and purification of CBD-tagged proteins to ensure correct folding and so, allow effective solid-phase immobilisation while maintaining protein function.


Silica immobilised PETase biodegradation

Through the data we collected we can show that the fusion of a silica-tag to the PETase variants did not reduce PETase activity but increased their activity. The silica tags were proven to immobilise functional proteins onto the silica surface. However, the actual immobilisation of PETase on silica beads decreases the enzyme activity, but this cannot be blamed on the immobilisation step itself. When we immobilised a small amount of PETase onto silica beads, we found that the enzyme activity could be retained. We have a reason to believe that PETase immobilised on silica beads retains its activity and stability if the protein loading to each silica bead is well-defined.

Immobilized PETase activity assessment

We assessed the PETase mutants' activity based on para-nitrophenol-butyrate (pNPB) assay (Figure 2), since the PETases can be hydrolyse the pNPB into para-nitrophenol (pNP). When this hydrolysis occurs, it will create a yellow colour which can then be detected by a spectrometer. The stronger the colour change the stronger the PETase activity.

Figure 2. The immobilized protein sample activity result based on para-nitrophenol-butyrate (pNPB) assay. The figure presented the fold-change of immobilized protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Car9-Dou_PETase] to [Tri_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.2]. The fold changes of activity from [FAST_PETase] to [FAST_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.3]. The reaction system was set up with final volume 1ml in each Eppendorf tube, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). The absorbance was measured from the Spectrometer at 415nm. [Shuffle without Lv.1 plasmid.2] was the protein sample from the same batch of the empty SHuffle strain as Tri-PETase constructs. [Shuffle without Lv.1 plasmid.3] was the protein sample from the same batch of the empty SHuffle strain as FAST-PETase constructs. “U”is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The activity of [Shuffle without Lv.1 plasmid.2] is 4.42E-02 U/mg protein sample. The activity of [Shuffle without Lv.1 plasmid. 3] is 1.55 E-02 U/mg protein sample.

The [FAST_PETase-linker-L2NC] immobilised onto the celite545 silica beads had the highest enzyme activity even though it had one of the lowest rates of enzyme bound to the celite545 (See Results page). These results show that we have active FAST_PETase bound to silica beads. These silica beads could then be placed into water to degrade PET and be used as part of our PET biodegradation device.



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