Genetic code expansion of plastic-degrading enzymes for detection of microplastics

Abstract

Petrochemical plastics can degrade into the size level of microplastics. One source of microplastics is polyethylene terephthalate (PET or PETE). Enzymes used to degrade PET have been reported. In our work, we wish to repurpose the activity of PET-degrading enzymes, into PET-detecting enzymes, and use the engineered enzyme for microplastic detection, via enzyme engineering to create enzyme variants which can form covalent adducts with microplastic particles. The enzyme-microplastic adduct is generated via the use of 2,3-diaminopropionic acid (DAP) derivative, which we will incorporate in place of the catalytic serine residue at the active site of I. sakeinesis—PETase via the genetic code expansion technique. Working toward this goal, we have successfully synthesized 2,3-diaminopropionic acid (DAP) derivative. The synthetic methods were carried out with improvements to the protocols previously reported in literature, in seven steps and an overall yield of 7 %. The compound was structurally characterized by nuclear magnetic resonance (NMR) spectroscopy. Moreover, we cloned an expression vector for PETase, in which its catalytic serine codon is replaced with an amber stop codon—marking it as a site for unnatural amino acid incorporation and demonstrated the expression of the PETase protein bearing an unnatural amino at the active site serine in Escherichia coli using amber-suppressor pyrrolysyl tRNA and its corresponding pyrrolysyl-tRNA synthetase enzyme.