Being a main goal of BIZENTE, partner EvoEnzyme has been constructing mutagenic libraries of enzymes and analyzing individual engineered variants with improved activity on model molecules. Model molecules of the four selected resins (Hexflow®RTM6, Araldine®LY1568, DION®IMPACT9102 and R930A) were used as substrates on high-throughput screenings after several libraries were functionally expressed in S. cerevisiae.
Figure 1. Representation as a 3D structure of a mutated variant of a resin degrading enzyme.
A group of ligninases and hydrolytic enzymes were detected with improved properties and presented mutations on different areas of the protein. The approach for the design of the libraries included general error-prone amplification of parental genes as well as focused methodologies that targeted specific fragments of the enzyme based on structure-function studies (Fig 1). These new variants are being used as parental types in new engineering processes to finally obtain the best candidates adapted for the different degradation processes that will be scaled-up on industrial settings.