From the beginning of BIZENTE, partner EvoEnzyme has been studying the potential of ligninolytic enzymes in the degradation of complex thermoset composites. With the aim of customizing these enzymes (by protein engineering) and adapt them to industrial settings, EvoEnzyme started testing their activity in the degradation of soluble model molecules based on the structure of four selected resins (Hexflow®RTM6, Araldine®LY1568, DION®IMPACT9102 and R930A).
For this task, several methods were developed based on the detection of changes in the absorbance of the reaction medium when the enzyme is incubated with the designed molecules (Fig. 1). Therefore, the oxidoreductase candidates to be engineered were selected according to the most promising colorimetric responses in presence of this model scaffolds for the resins. Additionally, these methodologies were adapted to high-throughput scenarios to allow the analysis of thousands of variants from a mutagenic library of enzymes. Once the screening protocols were set up, several mutagenic libraries based on error-prone DNA amplification were constructed and analyzed (Fig. 2).
|Figure 1. Colorimetric screening for modification model molecule based on Araldite LY 1568 (epoxy resin). Linearity for the assay was tested with increasing amounts of ligninase supernatants in the reaction (A). Colorimetric response with different reaction times (0, 15 and 30 minutes) (B).|
|Figure 2. Mutagenic landscapes for selected ligninase constructed with different DNA polymerases.|
You can also learn more about the activity of enzymes on low molecular-weight model compounds of representive thermoset composites in this article.