Latest revision as of 14:41, 21 October 2019

Coding sequence for S245I/R280L IsPETase double mutant

A coding sequence for mutated S245I/R280L PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.

A coding sequence of the PETase double mutant S245I/R280L.

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 analyze 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 residues and Leucine 288 are located at the corresponding positions of Serine 245 and Arginine 280 in subsite II of IsPETase.

The resulting double 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.

After the protein is induced using 0.5mM IPTG, it is purified and extracted using a column with nickel resin due to a 6X His-Tag fused with PETase outside the globular structure. After purification, SDS-PAGE can be performed to confirm successful expression.

Figure 1: SDS-PAGE of purified S245I/R280L double mutant of PETase. 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 is 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 wild type and S245I/R280L PETase. It can be seen that S245I/R280L has a lower optical density than the wild type, indicating lower activity

Figure 3: Percentage increase of optical density for reaction mixtures with wild type and S245I/R280L 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

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Sequence and Features