The overall goal of this project is to develop a platform for efficient plastics degradations. We combine enzyme bacterial surface display technology with high throughput kinetic assays to reduce costs and time associated with plastics degrading technology development and measurement assay. To achieve this goal we demonstrated the success of surface display enzymes and the ability of the kinetic assays to measure plastics degrading enzyme activities. Completing both of these goals proved a significant reduction in time and cost by eliminating purification steps and shortening enzyme characterization times.

As our project focuses on PET degradation, we used the enzymes PETase and MHETase as our enzymes to be surface displayed using transporter proteins YeeJ and AIDA-I. We were able to demonstrate activity with MHETase in the form of both YeeJ and AIDA-I based on standard degradation assay as shown in Figure 1. Although we found the limitation of the assay being unable to distinguish between intracellular and surface displayed MHETase, we are planning to figure out the display efficiency. Using a high activity variant of PETase called FAST-PETase, we also demonstrated activity for AIDA-I-FAST-PETase as shown in Figure 2 and we further plan to address the questions of surface display efficiency from observed autolysis after several days in storage.

Figure 1. HPLC measurements of OD 20 whole cell MHETase constructs after 168 hours of reaction at 25 °C. Reaction medium is 250 μM MHET in 50 mM Bicine-NaOH pH 8.5. (All same scale)

Figure 2. HPLC measurements of Pure FAST-PETase and OD 30 whole cell FAST-PETase constructs after 213 hours of reaction at 25 °C. Reaction medium is 50 mM Bicine-NaOH pH 8.5.

For the kinetic assay development, two processes were designed and tested. The BHET plate assay used a suspension of BHET, a material degraded by PETase, in an agar plate to qualitatively evaluate PET degrading activity. Despite its success in differentiating between active and inactive PET degrading enzymes, it was unable to distinguish between intracellular and surface displayed whole samples. The other kinetic assay used fluorescein dibenzoate (FDBz) as a substrate that would produce fluorescein for a quantifiable measurement of activity. This assay was also successful in illustrating increased activity for FAST-PETase compared to PETase as shown in Figure 3. Being able to quantify increases in activity for improved PET degrading enzymes is one of the key goals of the assay, as it is needed to replace or supplement the standard degradation assays. However, the FDBz assay gives unexpected results when used with whole cell samples, requiring troubleshooting before moving forward. Despite these limitations, both assays were shown to be completed in a matter of hours compared to the several day requirement of standard degradation assays, saving significant characterization time.

Figure 3. Relative fluorescence of PETase and FAST-PETase after 3 hours of reaction with 250 μM FDBz (0.5 μM purified enzyme, 37 °C)

We believe that the results discussed show promise for achieving the overall goal of the project with further work. We were able to show that our surface displayed constructs were active and that the kinetic assays developed are capable of rapid qualitative and quantitative measurements of activity on an improved PET degrading enzyme. Therefore, this serves as a proof of concept for the continued improvement of this project as we work to verify successful surface display and troubleshoot problems in kinetic assays.