Part:BBa_K2110104

PETase site-directed mutant R90A

This is one of the site-directed mutant of the PETase gene, we change the 90th amino acid from R to A. The module is the Saccharomyces cerevisiae codon optimization version of PETase gene. We use overlap PCR to obtain this mutant.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 79
Illegal BglII site found at 289
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Results

Rational Design

Since PETase was found to contain a GWSMG motif in accordance with the GXGXG motif^[2], which is characteristic and highly conserved in α/β hydrolase fold family, we simulated a best fit model for PETase by SWISSMODEL with a template as Thc_Cut2. As expected, the homology model of PETase displays a canonical α/β hydrolase fold with a Ser¹⁶¹-His²³⁷-Asp²³⁷ catalytic triad and a preformed oxyanion hole (Fig.1), suggesting a classic serine hydrolase mechanism.

In addition, we generated a multiple sequence alignment by ClustalX with seven serine hydrolases from NCBI conserved with GXSXG motif and catalytic triad and reported to be able to degrade PET (Table.1).Based on the mutiple sequence alignment result(Fig.3), we found the 90th (with signal peptide excluded) amino acid residue arginine is quite conversed as threonine in other seqences.

According to the 3D stucture, the 90th amino acid is on the surface of protein and rather adjacent to binding site residue Tyr⁸⁷. As arginine is the most hydrophilic amino acid of relativley huge volume, we decided to subsitute it with threonine(conserved residue) and alanine. As alanine is much smaller than arginine and is also a more hydrophobic amino acid, the following result indicated an improved hydrolysis activity of mutant R90A compared to the wild-type enzyme.

Assay system—CFPS system

We detect the absorption peak of culture medium after culturing with PET film added for 2d. The absorption degree-wavelength curve is as follows.

We used the relative fluorescence units to represent the expression level of enzymes.

Finally we got the relative activities of enzymes.

Reference

[1]Shaorong Chong. Overview of Cell-Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications. Current Protocols in Molecular Biology 16.30.1-16.30.11, October 2014 DOI: 10.1002/0471142727.mb1630s108

[2] Chen S, Tong X, Woodard R W, et al. Identification and characterization of bacterial cutinase[J]. Journal of Biological Chemistry, 2008, 283(38): 25854-25862.