Latest revision as of 00:40, 22 October 2019

PETase Reconstructed Ancestor 1

An important part of our project is engineering more stable enzymes that will last longer in our filter. In order to achieve this, we decided to make use of the method of ancestral reconstruction. This technique is used to recover ancestral traits that are useful but have been lost during the process of evolution; and relies on a large number of sequences, phylogenetic analysis, and modelling.
We started by searching current papers for phylogenetic trees of PETase that have already been built. We identified a paper that had already organised plastic-degrading enzymes into categories, and had identified a set of enzymes that were closely related to PETase. We used BLAST software to search for sequences homologous to PETase and the set of enzymes previously identified in the paper; and through this process identified 243 homologous sequences. These sequences were aligned and narrowed down to 76 sequences with the help of Professor Harmer from the University of Exeter Living Systems Institute. The final alignment of the sequences was then fed into the ANCESCON software that performed the ancestral reconstruction. The software produced a phylogenetic tree and identified 74 ancestors. In order to identify the most suitable ancestors to model and use, we used the method below to weight each one:
Following this method, we identified 4 potentially suitable ancestors to further analyse. The YASARA software was used to model the 3D structure of each of the four ancestors. The models produced were then aligned against the structure of PETase by Professor Harmer in order to identify significant changes in the sequences. We discovered that the catalytic triad was conserved in all four ancestors, suggesting that the PET degrading activity had not been lost. Interestingly, we have also discovered that a beneficial mutations reported in past papers was already present in all four of the ancestors, namely R280A. The only significant trait lost during the reconstruction was the second disulfide bond that was present in PETase but not the ancestors. However, we reverted this by changing the two alanine residues in the ancestors with two cysteine residues. Additionally, we deleted the first five amino acids from the N-terminus that we suspected were composing the signal peptide. Once these minor adjustments had been completed, we sent the final sequences to be synthesised.

Characterisation

In order to characterise our part and determine the rate of its activity and prove its functionality we have run a series of experiments. After transforming the Arctic Express, Rosetta Gami and BL21 DE3 strains of E. coli with our plasmid we induced the expression of the enzymes using IPTG. In order to confirm that the enzyme expression has been successful we ran a western blot which showed the presence of the enzyme in the soluble fractions of the sonicated cells. Afterwards the enzyme was purified and used in assays to show its functionality and determine the rate of its activity.

Expression in E.coli

Western blot of the soluble fraction of Arctic Express strain showing expression of all Ancestral reconstruction mutants. The PageRuler Plus prestained protein ladder was used and labelled with the corresponding sizes. The negative control is in lane 1. WT PETase is in lane 2. Ancestral Reconstruction Mutant 1 is in lane 3. Ancestral Reconstruction Mutant 2 is lane 4. Ancestral Reconstruction Mutant 3 is lane 5. Ancestral Reconstruction Mutant 4 is lane 6.

Purification graphs

Nickel Affinity Chromatography

Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 48 ml shows protein elution.

Size Exclusion Chromatography (Superdex-75)

Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 column.

Esterase Activity

Activity was measured by spectrophotometrically flowing the hydrolysis of p-nitrophenyl acetate (pNPA) into acetate and p-nitrophenol. This was performed at room temperature in buffer containing 50 mM NaPhosphate buffer pH7.5, 100 mM NaCl. A range of substrate concentrations were tested and a blank used to subtract the auto-hydrolysis of the pNPA. The production of p-nitrophenol was measured at 405 nm.

The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations.

The specific activity of the enzyme at differing substrate concentrations.

Thermal Stability Graphs

Thermal Stability

The thermostability of the enzyme was investigated incubating enzyme samples at a range of temperatures (20 °C - 90 °C) for one hour using the gradient function in a SensOQuest LabCycler (Geneflow) before samples are cooled to 4 °C and assayed for activity using the esterase assay method described previously.

The thermal stability assay shows the production of p-nitrophenol (A405nm) after the pre-incubation of the enzyme at increasing temperatures before the esterase assay was carried out.

Thermal Stability of BBa_K3039017 (AP1) Vs. Wild Type PETase

The percentage activity compared to the enzyme activity at room temperature.

Conclusion

The ancestral mutants were cloned and over expressed in E.coli and did show esterase activity. Although AR1 is unable to retain as high a level of activity at some of the lower temperatures AR1 is able to retain ~25 % activity at 50 °C. This is an improvement on the WT PETase which shows no activity at this temperature.

References

[1] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.

[2] Keiko Watanabe, Takatoshi Ohkuri, Shinichi Yokobori, Akihiko Yamagishi; Designing Thermostable Proteins: Ancestral Mutants of 3-Isopropylmalate Dehydrogenase Designed by using a Phylogenetic Tree (2006) J. Mol. Biol. 355(4), 664-674

Sequences and Features