Latest revision as of 00:39, 22 October 2019

PETase R208A

Usage and Biology

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles.

This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the R208A mutant of PETase, with an N-terminal His-tag. The His-tag was added to allow for conformation of expression and subsequent purification. This mutation has been reported in the literature to increase the activity of PETase (Seo et al 2019).

The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, to allow for potential cloning into alternative BioBrick plasmids. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 21 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.

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 to determine its rate of activity.

Expression in E.coli

Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase R280A) is labeled with 5. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.
Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase R280A) is labeled with 5. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Protein Purification

Nickel column

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 54 ml shows protein elution.

Size Exclusion Column (Superdex-75)

Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 column. While the trace is quite messy the largest peak with an elution volume of ~80 ml shows the monomeric form of the protein being eluted from the 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

Conclusion

The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. This enzyme was the worst expressing enzyme from the rational design section. The enzyme had the worst specific activity when compared to the WT PETase (0.3 x).

References

[1] Seongjoon Joo, In Jin Cho, Hogyun Seo, Hyeoncheol Francis Son, Hye-Young Sagong, Tae Joo Shin, So Young Choi, Sang Yup Lee & Kyung-Jin Kim; Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation (2018) Nat. Commun. 9(382)

Sequences and Features