Part:BBa_K808025

FsC: Cutinase PET cleaving enzyme

A cutinase is a cuticula degrading hydrolase from the fungus Fusarium solani pisi. It shows activity towards PET.

Usage and Biology

The characterization of FsC occured in 3 different ways.

Our [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Simulation Simulation Lab] performed

• Docking Simulation

Our [http://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science Material Science Lab] performed

• Surface analysis of polyethylene terephthalat with atomic force microscopy

Our [http://2012.igem.org/Team:TU_Darmstadt/Project/Degradation Degradation Lab]

• Enzyme Activity assay

Docking Simulation

[http://2012.igem.org/Team:TU_Darmstadt/Modeling_Docking#Docking Docking Simulations]

Figure 1: Homology model of Fusarium solani cutinase, special thanks and regards to our Simulation Lab

We extracted binding energy from the atomic B-factors and the binding constant from the atomic property derived from all our calculations. We used scatter plots with the binding constant and the inhibition constant in order to compare the plasticizer as well as the PET substrate. We compared the binding energies as well as the inhibition constant from the best ranked docking runs.

Here, OET (dimeric state) became our reference substrate, meaning if an additive exhibits a higher inhibition constant it may effect our protein. Otherwise, if a binding energy exhibits a higher level than OET we can conclude that an additive may accumulate on our protein surface. Moreover, we compare the binding as well as the inhibition energies of the additives with itself to identify the strongest binder. Since we know the active site of the Fs. Cutinase and the PnB Est. 13, it will be interesting if a plastizicer may dock there. In order to analyze that, we counted the residues which are involved within every docking simulation. Therefore, we measured the distance of every ligand to its corresponding receptor with an appropriate threshold and thus identified the residues.

For the sake of convenience of this page, please visit our [http://2012.igem.org/Team:TU_Darmstadt/Modeling_Docking#Docking Simulation Lab > Docking Simulation] for further informtion.

Surface analysis of polyethylene terephthalat with atomic force microscopy

[http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Material_Science#Surface_analysis_of_polyethylene_terephthalate_with_atomic_force_microscopy_.28AFM.29 Surface analysis of polyethylene terephthalat with atomic force microscopy]

Atomic force microscopy is used to make very precise surface analysis up to nanometer scale. The goal was to indentify differences between different modifications of polyethylene terephthalate and to proof enzymatic degradation by changed surface properties.

Experiment 1

A piece of a PET water bottle is melted between two metal plates using a heat gun to create a flat sample. It is incubated for 24 h at 37 °C after applying a solution of 90 µmol/L FsC. The Sample is washed with distilled water and dried in a cabinet dryer at 60 °C for 1 h. The samples are investigated using AFM. Surface modifications of PET induced by FsC could be observed in form of circular perforations. Measurements in this area show a surface profile of (±100 nm). Measurements of the reference show a surface profile of (±30 nm). The pictures below show surface profiles and light microscopic captures of the non treated PET reference and the FsC treated PET surface.

AFM of PET surface after FsC exposure:

AFM of the PET surface as reference:

Experiment 2

Pieces of PET foil were added to a 2 µmol/L solution of FsC. The solution was constantly agitated in 50 mL falcon tubes. After 7 days the samples were washed with distilled water and dried in a cabinet dryer at 60 °C over night. Afterwards the samples are investigated using AFM. FsC induced surface modification of PET could be observed. The Pictures below show 3D models, height profiles and the cantilevers amplitude as well as the phase deviations. The 3D models give a first impression of the texture of the surface. Looking at the height profile and the amplitude deviation more detailed informations about the surface texture can be obtained. The reference samples show a very flat surface (±3 nm). Bigger deviations are due to manufactionary mistakes of the foil. FsC treated surfaces show a deviation ten times bigger than non treated ones (±30 nm). Comparing the phase deviation of FsC treated surfaces with the reference, there can be seen big diferences due to varying mechanical properties. These are caused by degradation by FsC. The Polymer chains are cut in to smaller pieces which then stand out of the surface, letting it swell. The resulting surface is not only rougher but persumably less dense and therefore less firm.

AFM of PET foil after FsC exposure :

AFM of PET foil as reference :

Enzyme Activity Assay

[http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Enzyme_Activity_Assays Enzyme activity assay]

pNP-Assay

We used a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#pNP-Assay pNP-Assay] in order to describe.

Conditions were set at: T = 34°C and pH = 7.4. The method is simple. The absorption is measured every minute over 30 minutes by an ELISA reader capable of 96 well plates, after addition of enzyme. The released pNP-group has a typical absorption at 405nm wavelength.

The mechanism of pNPB degradation. The release of pNP is measured by the characteristic absorption at 405 nm

Lineweaver-Burk plot of the enzymatic activity of FsC. The kinectics were quantified by measuring the absorbtion at 405 nm over time. 405 nm is the characteristic absorption of pNP, being released due to hydrolytic activity of FsC

K_m and V_max calculation

To find K_m the growth of the first twelve data points were written down in another diagram. The slope of the regression line gave the speed of the hydrolysis for the single substrate concentrations. Now the value of the speed with the associated concentration of substrate were plotted in a Lineweaver-Burk diagram. At the point where the regression line is crossing the x-axis the x-coordinate amounts to -1/K_m. So K_m is about 400µM. To calculate the value of V_max we used the point where the plotted line crosses the y-axis. The associated y-coordinate is 1/V_max. Consequently V_max for 5nM FsC is 0,001467 1/s.

K_cat calculation

The K_cat value is calculated through dividing V_max by the enzyme concentration of 5nM. K_cat counts 0,293395 1/mol*s.

Indicator Assay

For a more visual proof of enzymatic activity we used Monomethyl terephthalate as a substrate and FsC as a catalyst. We used [http://en.wikipedia.org/wiki/Bromothymol_blue bromthymol blue] as an indicator. The release of COOH groups, due to hydrolytic degradation by FsC results in a colometric change from green to blue. For a more detailed description please visit our [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Degradation_of_monomethyl_terephthalate_over_time labjournal> Degradation of monomethyl terephthalate]

Principle of degradation of monomethyl terephthalate.

Day1: Degradation of monomethyl terephthalate. Left: with Est13(~0,8µM) Middle: with FSC(~8µM) Right: Negativ sample

Day8: Degradation of monomethyl terephthalate. Left: with Est13(~0,8µM) Middle: with FSC(~8µM) Right: Negativ sample

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 379
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 525
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 301