Identification and expression of a novel urethanase enzyme and development of a urethanase assay

Abstract

Plastic recycling is a difficult process because of the variability in starting material and contamination with food and other chemicals. Very little of current plastic production comes from recycled material. In addition, many plastics are sourced from petroleum, which is nonrenewable and releases toxic chemicals into the environment. However, more plastic is produced each year. To solve this problem, it is necessary to devise new methods for recycling plastic. In this thesis, two bacterial isolates are shown to grow using polyurethane foam as their sole carbon source, their genomes sequenced, and proteomics methods used to identify potential urethanase enzymes. Next, enzymes were expressed in E. coli and tested using an Impranil assay. Finally, two novel urethanase assays were developed, a fluorometric one using a synthesized urethane substrate, and a mass-spectrometry one directly using particles of polyurethane foam. Genome sequencing of the two isolates produced assembled draft sequences, one of which was fully polished and circularized, and the other which was polished but did not circularize. Proteomics identified a large number of hydrolase enzymes overexpressed when isolates were grown in polyurethane media, of which nine were selected as likely urethanase candidates. One enzyme was successfully expressed, but it was a slightly truncated variant and only expressed in low quantities. All three enzyme assays were tested and resulted in promising proof-of-concept results