Difference between revisions of "Team:UMaryland/TPAvPCA"

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We ran the simulation with the following parameters:
 
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Cellulose density = 1.5 g/cm3
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- “600 uM” of PET added in solution
 
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Total working weight of cellulose = (1.5 g/cm3)(322π cm²)(0.01 cm) = 15.87  g cellulose
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- Constant enzyme concentrations
 
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Using values obtained from the literature about the CipA-CBD [1] where our CBD was acquired from, we determined:
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- Reversibility of reactions not considered
 
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Maximum binding of CipA-CBD to cellulose = 0.43 µM/g cellulose
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- Simulated rich oxygenated media with excess NADPH
 
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Local concentration of PETase at 50% binding = (0.5)(15.87 g)(0.43 µM/g) = 3.41 µM PETase
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- Fixed 50 uM of transcription factor concentrations (a gross exaggeration)
 
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Enzyme Kinetics
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- 30 Day simulation
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The degradation of PET follows heterogeneous catalysis, where the reactants and the products [2] are in different phases. This creates difficulty in modeling the rate of degradation but teams such as Tianjin China has used Langmuir's Equation, secretion, and diffusion models to model the process of secretion, diffusion, adsorption, and degradation. However, while their models gave great insight into the mathematical relationships between these variables, it was not possible to obtain practical results as many of the variables used in the equation are not found in the literature and are difficult to obtain experimentally.
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We have developed the “working cellulose” model which is more practical in predicting the rate of degradation when PETase with a cellulose binding domain is added to a degradation system. We modeled the rate of degradation of PETase as a zero-order system, where the concentration of the reactant is negligible to the rate of the reaction. This is present in many surface-limited chemical catalysis models, where the catalyst surface is saturated by the reactants, and the rate limiting step can be simplified in the surface area of the catalyst.
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Simulation Results
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However, in our PET degradation model, the reverse holds true, where the concentration of PETase is the rate limiting variable compared to the surface area of the PET film when considering practical examples such as the degradation of a plastic bottle. We determined the following rate equation for the degradation of PET:
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In the PCA detection system, we predict that there are high levels of PCA in the media, but the rate limiting reaction in the degradation pathway is the conversion of DCD to PCA. Transport of PCA limits the activation of the PcaU. The model predicts that concentrations of PCA will exceed the solubility limit (120 uM in water), but we see that there is full activation of PcaU prior to the solubility limit being reached. There is rapid activation of PcaU at about 1.5 days.
 
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In our reduced and simplified zero order enzyme kinetics equation, we only need to know the product, time, and enzyme concentrations to determine the kinetic variable k to predict degradation. Through values obtained through the original PETase discovery paper by Yoshida et al, we were able to calculate the kinetic variable for both low-crystalline and high-crystalline PET.
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In the TPA detection system we see an immediate increase in the concentration of TPA in the media, with a similar problem of the rate of transport limiting the activation of TphC. In this simulation, we see an increase in the time to full response to about 7 days, which is about 4x longer than the PCA based system. However, in this simulation we exceed the solubility limit of TPA (90 uM) rapidly, therefore we tweaked the model to a fixed concentration 90 uM concentration of TPA in the media.
 
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At 30°C, pH 7.0, [PETase concentration] = 50nM, time = 18 hrs: PETase produced a total of 0.015 mM of product when fed high-crystalline PET and 0.3 mM of product when fed thin-sheet PET.
 
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Using above data we can determine the rate constants.
 
  
 
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Results
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Therefore, with the calculated local concentration of our enzyme and our enzyme kinetic parameter we were able to predict the rate of degradation of a plastic water bottle. If we desire 4 grams of PET to completely degrade to its subsequent product MHET, using the molecular weight of MHET (Mw = 210.185 g mol-1), we determine that the final concentration of MHET in a 1 L container would be 0.019 M. Since PET is not an aqueous substance, it is difficult to assign it a “concentration” value for our equation. Therefore we would determine the creation time of MHET rather than the degradation time of PET.
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Using our equation:
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With the following parameters:
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-  [PETase] = 3.41 µM
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-  k = 0.00463 s-1 for high crystalline PET
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-  [MHET] = 0.019 M
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Solving for time, we predict that complete degradation of one PET bottle sandwiched between two bacterial cellulose sheets with 50% saturation of our PETase-CBD fusion protein will occur in 1.2 x 106 seconds, or approximately 14 days.
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As degradation progresses, the appropriateness of our zero-order enzyme kinetics model will decrease as the surface area of PET available to our enzyme will become more significant. Therefore, a time to 50% degradation will be more indicative of the actual degradation of PET. We determined this value to be about 7 days.
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Revision as of 13:16, 17 October 2018

Template Title Template Title

Bacterial Cellulose
Does the addition of a cellulose binding domain accelerate the degradation of PET?
When we ran into the issue of detection of the degradation of PET, we found two potential sensor systems that we would be able to use, one which detected protocatechuate (PCA) and the other detecting terephthalate (TPA). To determine which sensor system was better for our needs, we used the MatLab SimBiology package to model the degradation of PET down to PCA and further to the cellular metabolite. The SimBiology files are available at this link:
Simbiology Zip File for TPA vs PCA
Simulation Setup
There are four major processes that occur in our sensor setup as shown below:
Waluigi Time!
which are the depolymerization of PET into TPA, the degradation of TPA to PCA, the transport of PCA and TPA, and the expression of GFP from the activation of transcription factors. The parameters of the TPA and PCA sensor systems are described below:
Waluigi Time!
Using the SimBiology we created two sensor systems for our degradation analysis:
Waluigi Time!
Through an extensive literature search, we found the enzyme kinetics parameters and protein concentrations as listed below. We converted all values of Vmax and kcat in units of M product / (s * M protein). We used non-reversible Michaelis-Menten as our enzyme kinetics parameters except the degradation of PETase, which we approximated using our zero order enzyme kinetics equation described previously and the transport of TPA and PCA by TpiAB and PcaK respectively, which followed a law of mass action kinetics.
Waluigi Time!
We ran the simulation with the following parameters:
- “600 uM” of PET added in solution
- Constant enzyme concentrations
- Reversibility of reactions not considered
- Simulated rich oxygenated media with excess NADPH
- Fixed 50 uM of transcription factor concentrations (a gross exaggeration)
- 30 Day simulation
Simulation Results
Waluigi Time!
In the PCA detection system, we predict that there are high levels of PCA in the media, but the rate limiting reaction in the degradation pathway is the conversion of DCD to PCA. Transport of PCA limits the activation of the PcaU. The model predicts that concentrations of PCA will exceed the solubility limit (120 uM in water), but we see that there is full activation of PcaU prior to the solubility limit being reached. There is rapid activation of PcaU at about 1.5 days.
Waluigi Time!
In the TPA detection system we see an immediate increase in the concentration of TPA in the media, with a similar problem of the rate of transport limiting the activation of TphC. In this simulation, we see an increase in the time to full response to about 7 days, which is about 4x longer than the PCA based system. However, in this simulation we exceed the solubility limit of TPA (90 uM) rapidly, therefore we tweaked the model to a fixed concentration 90 uM concentration of TPA in the media.
1: Sabathé, F. & Soucaille, P. Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum. J. Bacteriol. 185, 1092–1096 (2003).
2: Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).

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