Part:BBa_K2091005

LC-Cutinase Variant F4

This is a LC-Cutinase mutated protein. It has 4 amino acid mutations compared to the codon optimised protein. The mutations are not restricted to distance from the active site of the protein.

Figure 1: A 3D model of the F4 protein.

Characterization

pNP-Butyrate degradation assay

In order to evaluate the activity of the F4 mutant LC-Cutinase we used a pNP-Butyrate degradation assay (see Protocols *LINK*).
At first we used different concentrations of the substrate - 50/125/250μM and measured the absorbance at 405nm from the moment the enzyme was added.

Figure 2: pNP-Butyrate degradation activity of all LC-Cutinate variants and W.T. at a substrate concentration of 50μM. For control we used E. coli strain BL-21 without any vector ("BL-21") with pACYC plasmid backbone only ("pACYC").

Figure 3: pNP-Butyrate degradation activity of all LC-Cutinate variants and W.T. at a substrate concentration of 125μM. Controls are the same as with 50μM concentration.

Figure 4: pNP-Butyrate degradation activity of all LC-Cutinate variants and W.T. at a substrate concentration of 250μM. Controls are the same as with 50μM concentration.

After determining an optimal substrate concentration we measured the activity of each mutant variant with a substrate concentration of 125μM and different enzyme concentrations.
Protein concentrations were determined with spectrophotometric methods after purification with a cation exchange column.

Figure 5: pNP-Butyrate degradation assay results of the LC-Cutinase F4 variant compared to the W.T protein.

After receiving O.D values above 1 we lowered the starting concentration of pNP-Butyrate to 75μM and measured the activity again. We received a linear trend from the start of the measurement, suggesting our variant is very fast at degrading the substrate.

Figure 6: pNP-Butyrate degradation assay results of the LC-Cutinase F4 variant using different concentrations.

PET degradation assay

The LC-Cutinase variants were grown on M9 minimal medium plates with shredded PET pellets as a sole carbon source to test their ability to degrade PET.

Figure 7: LC-Cutinase F4 variant grown on a M9 agar plate with shredded PET pellets as a sole carbon source. Colonies are marked in red.

Figure 8: Control. E. coli expressing the W.T LC-Cutinase protein grown on a M9 soft agar plate with no carbon source.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]