Expression and engineering of unexplored PET degrading enzymes from <i>Microbispora, Nonomuraea, Micromonospora</i> genus

ABSTRACT Low recycling rates have resulted in the alarming rate of accumulation of a widely used plastic material, polyethylene terephthalate (PET). With the build-up of plastics in our environment, there is an urgent need to source for more sustainable solutions to process them. Biological methods such as enzyme-catalyzed PET recycling or bioprocessing are seen as a potential solution to this problem. Actinobacteria, known for producing enzymes involved in the degradation of complex organic molecules, are of particular interest due to their potential to produce PET degrading enzymes. The highly thermostable enzyme, leaf-branch compost cutinase (LCC) found in Actinobacteria is one such example. This work expands on the discovery and characterization of new PET degrading enzymes from Microbispora, Nonomuraea , and Micromonospora genus. Within this genus, we analyzed enzymes from the polyesterase-lipase-cutinase family, which have ∼60% similarity to LCC, where one of the enzymes was found to be capable of breaking down PET and BHET at 45-50 °C. Moreover, we were able to enhance the enzyme’s depolymerization rate through further engineering, resulting in a two-fold increase in activity. IMPORTANCE The proliferation of PET plastic waste poses a significant threat to human and environmental health, making it an issue of increasing concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Despite the existence of enzymes capable of degrading PET, their scarcity in nature limits their applicability. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel ones that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation.