Latest revision as of 14:24, 20 October 2021

Coding sequence for S245I IsPETase double mutant

A coding sequence for mutated S245I PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.

A coding sequence of the PETase single mutant S245.

This sequence is modified from a sequence for wild type PETase which was also codon optimised for Escherichia coli, obtained from previous studies done on PETase.[1]

Origin and biology

The enzyme is a hydrolase which degrades polyethylene terephthalate into simple molecules: MHET, BHET, and TPA by cleavage of the ester bond within the polymer. It was originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.

Design

We analyzed the rationale for PETase mutant design from previous studies done on the residue modification of this enzyme. A clear trend in most successful mutation attempts is that an increase in hydrophobicity or a binding site similar to T. fusca cutinase, which is narrower.

The mutation sites for this mutant are located in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction.

In TfCut2, Isoleucine 253 residue is located at the corresponding positions of Serine 245 in subsite II of IsPETase. The resulting mutant makes the substrate binding site, subunit II more cutinase-like and increases the hydrophobic property of the enzyme.

Characterisation

In our experiments, to insert this gene into cells, the PET-21b vector is used due to its high copy number and the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into C41(DE3) cells, which we use to perform the protein induction due to the toxic nature of PETase.

Figure 1: SDS-PAGE of purified PETase single mutant. A band of around 30 kDa is clearly shown

As shown above, the thick band around 30 kDa shows successful expression of the construct. After protein purification, an enzyme assay can be performed to confirm the protein activity.The substrate used is p-nitrophenyl dodecanoate, as the ester bond is similar to that in PETase. The product, p-nitrophenol has a yellow colour. Therefore, activity can be confirmed by measuring optical density at 415nm.

Figure 2: Optical densities at 415nm for reaction mixtures with S245I and Wild Type PETase

Figure 3: Percentage increase of optical densities for reaction mixtures with S245I and Wild Type PETase

As shown, there is a clear increase in optical density, confirming enzyme activity.

[1]:Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

SEM & HPLC data

We are team iGEM21_HK_GTC.
The authors of this contribution are Tam Ching Yeung, Leung Gabriel and Wong Chun Hei.
In the existing part BBa_K2982005, we added Scanning Electron Microscope (SEM) photos of PET films after being digested by S245I PETase mutant. The usage of SEM can accurately visualize how serious the PET film is digested by the enzyme.

(A) SEM images of digested PET films

We performed PET film digestion to study the PET hydrolytic activity of S245I PETase. After incubating 9μg of purified proteins with PET film at 30°C for 96 hours, we viewed the digested PET film under a SEM.

The pitting resulting from the digestion of S245I PETase mutant can be visualized, and the buffer-only SEM photo acts as a control.

(B) HPLC profile of the products released from the PET films

HPLC profiles demonstrated that the detection peaks representing TPA monomer and MHET intermediate product formed during PET film digestion have retention times at 4.64 minutes (Figure 2a) and 5.17 minutes (Figure 2b) respectively.
HPLC profiles of products released from PET film digestions using S245I PETase revealed incomplete PET hydrolysis as considerable amounts of intermediate product, MHET which showed a peak at 5.07 minutes were detected (Figure 2c).
Therefore, we can conclude that S245I PETase exhibits hydrolytic activity for PET depolymerization.

Sequence and Features