Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants.

Ancestral sequence reconstruction for protein engineers.

In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families.