Team:HK GTC/Design
Project Design
Aim of project
We aimed to enhance the performance of PETase by changing the properties of amino acid residues in the substrate binding site.
Rationale of mutant design
We mainly referred to five scientific papers that investigated the function and mechanism of PETase[1-5], and four of them performed site-directed mutagenesis of PETase. We went through their experimental processes, looked at their rationale of mutant design, collected and analyze their results.
From the findings of scientific papers, overall structure of PETase can be divided into different domains including substrate binding site (subunit I, subunit IIa, subunit IIb and subunit IIc), catalytic triad in the active site, cutinase-like active site cleft and disulphur bridges. Interestingly, most mutations performed in substrate binding site, preferably subunit II have a higher chance to succeed. Therefore, we gather a list of amino acid residues in PETase that have a high probability to interact with substrate in substrate binding sites.
Besides PETase, some other enzymes such as cutinases and lipases also show activities of degrading PET but as they are not specifically adapted for the function, they have low enzymatic activities. Among those enzymes, a cutinase from Thermobifida fusca which has close sequence identity to PETase, have the highest degradation activity Its substrate binding site is narrower than that of others. PETase exhibits a more open active-site cleft than homologous cutinases. Thus, it is hypothesized that making PETase’s substrate binding site narrower, into a cutinase-like structure could be a possible way to enhance its function.[1]
Furthermore, as PET molecules are hydrophobic in nature, it is hypothesized that an enzyme surface with increased hydrophobicity could interact with the substrate better and therefore increase the enzymatic activity. Therefore, we modified the potential amino acid residues to more hydrophobic amino acids which could lead to stronger hydrophobic interactions after mutation.
After examining all possible mutation sites, we came up with three double mutants and one single mutant, containing five mutation sites based on the aforementioned principles.
| Sites | Positions | Change |
|---|---|---|
| S245I | Near the end of the substrate binding site, subunit II where three MHET moeities are bound through hydrophobic interaction. | In TfCut2, Isoleucine 253 residues is located at the corresponding positions of Serine 245 in subsite II of IsPETase, The resulting single mutant makes the substrate binding site, subsite II more cutinase-like and increases the hydrophobic property of the enzyme. Thus, we hypothesized that single mutant of S245I may show enhanced PETase activity. |
| S245I/R280L | Near the end of the substrate binding site, subunit II where three MHET moeities 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. Thus, we hypothesized that double mutant of S245I/R280L may show enhanced PETase activity. |
| W159H/S245I | The mutation sites locate in substrate binding site, in subsite II where three MHET moieties are bound through hydrophobic interaction. | In TfCut2, Histidine 169 residues and Isoleucine 253 are located at the corresponding positions of Trpytophan 159 and Serine 245 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. Single mutant of S238F show decreased PETase hydrolytic activity while double mutants of S238F/W159H show improved activity[1]. Thus, we hypothesized that W159H may have synergistic effect with S245I which leads to an increase in enzyme activity. W159H/S245I double mutant can also be used to compare the PETase activity of S245I single mutant. |
| W159H/S214H | W159 locate in substrate binding site, in subsite II where three MHET moieties are bound through hydrophobic interaction. S214 locate in the edge of the substrate binding site have effect on substrate binding as it allows W185 wobbling for the binding of ligand. | Both single mutant of W159H and S214H show higher activity than that of the wild type enzyme[2]. Histidine is often found in the active sites of enzymes, where its imidazole ring can readily switch between uncharged or positively charged to catalyze the making i and breaking of bonds. In TfCut2, Histidine 169 residues and Histidine 224 are located at the corresponding positions of Trpytophan 159 and Serine 214 in subsite II of IsPETase, Thus, we hypothesized that the resulting double mutant may have synergistic effect on substrate binding. |
Our plasmid construct:
After finishing the design of the mutants, we modified the E. coli codon-optimised sequences from previous academic papers about PETase[1] for the constructs. These sequences were cloned into a PET-21b vector, which is often used in studies on PETase due to a high copy number and high gene expression level. For this vector, a lac operon is used for conditional expression. We used IPTG to induce the expression of PETase.
After finishing the design of the mutants, we modified the E. coli codon-optimised sequences from previous academic papers about PETase[1] for the constructs. These sequences were cloned into a PET-21b vector, which is often used in studies on PETase due to a high copy number and high gene expression level. For this vector, a lac operon associated with T7 promoter is used for conditional expression of PETase that fused with a 6XHis-Tag. IPTG is used to induce the expression of PETase.
One difficulty of expressing PETase in Escherichia coli is that it is a toxic protein for the bacteria. Causing declined cell growth or cell death if it is overexpressed and take up a significant portion of whole cell protein. Using conditional expression, we can prevent cell death caused by overexpression of the toxic protein.
Thus, we attempted to optimise a protocol for protein expression using IPTG in a small scale. We tried a range of IPTG concentrations ranging from 0 to 1mM. We also attempted to optimise a protocol for protein expression in a large scale. After expression, we isolated PETase from other cell proteins using a nickel column due to the presence of a 6xHis-Tag at the C terminal.
Then we used Bradford assay to determine the concentration of the enzyme produced.
After all of our mutants were successfully produced and isolated, we proceeded to test their activity. This was done by using an enzyme assay using p-nitrophenyl dodecanoate. This was due to it having an ester bond similar to that in PETase. When it is cleaved, the absorbance at 415nm of the reaction mixture increases due to para-nitrophenol being produced.
Using the enzyme assay, we measured the absorbance of reaction mixtures containing different mutants of PETase and compared their activities. We compared the results of our mutants with that of the wild type, and W159H/S238F, a successful mutant showing enhanced PETase activity made by experts in 2018.[1]
References:
[1]: Austin, H. P., et. al(2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115
[2]:Liu, B., He, L., Wang, L., Li, T., Li, C., Liu, H., Luo, Y. and Bao, R. (2018). Cover Feature: Protein Crystallography and Site-Direct Mutagenesis Analysis of the Poly(ethylene terephthalate) Hydrolase PETase from Ideonella sakaiensis (ChemBioChem 14/2018). ChemBioChem, 19(14), pp.1464-1464.
[3]: Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359
[4]: Han, X., Liu, W., Huang, J., Ma, J., Zheng, Y., Ko, T., . . . Guo, R. (2017). Structural insight into catalytic mechanism of PET hydrolase. Nature Communications, 8(1). doi:10.1038/s41467-017-02255-z
[5]: Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H., Shin, T. J., . . . Kim, K. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-1