We can't find the internet
Attempting to reconnect
Something went wrong!
Hang in there while we get back on track
Cell-free expression for enzyme discovery: Screening putative polyester polyurethane degrading enzymes with semi-automated cell free expression and nitrophenyl probes
Summary
Researchers used cell-free expression technology to rapidly screen bacterial enzymes for their ability to break down polyurethane plastics. Enzymes from biofilms found on aircraft were tested, identifying candidates that could potentially be developed for biodegradation of plastic pollution.
In recent years, Cell-free expression (CFE) has emerged as a valuable tool for the rapid prototyping of enzymes in discovery efforts. This study focuses on demonstrating the effectiveness of CFE in screening potential polyurethane degrading enzyme sequences obtained from biofilm samples collected from aircraft, followed by genomic analysis. To streamline the process and ensure consistent results, an automated fluid handling system using a low-cost Opentrons instrument was employed. This automated system allowed for the assembly of multiple 30 µL reactions in the screening process, while maintaining precise temperature control. A benchmarking comparison was conducted between the automated process and manual operation, revealing greater consistency and reliability with the automated protocol, particularly when incorporating pipette tip changes. In this study, a total of 13 putative hydrolase enzymes sourced from aircraft biofilm organisms, along with the previously validated polyester-degrading cutinase, were expressed using in-house E. coli extract and minimal linear templates. The expressed enzymes were then tested for activity directly within the extract, with the hydrolysis of nitrophenyl substrates measured through absorbance readings. The highest absorbance changes were observed for 4-Nitrophenyl Hexanoate and 4-Nitrophenyl Valerate. Through this screening process, two of the putative enzymes exhibited statistically significant activity above the baseline. This demonstrates the potential of CFE in conjunction with automated fluid handling to rapidly prototype and screen for polymer-degrading enzymes derived from environmental consortia. These findings signify the promising application of CFE and automated systems in the efficient identification and characterization of enzymes with the ability to degrade polymers.
Sign in to start a discussion.
More Papers Like This
Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes
Researchers used a rapid lab technique called cell-free expression to screen enzymes that might break down polyester polyurethane plastics, sourcing the enzymes from bacteria found growing on aircraft and vehicle surfaces. They identified 10 enzymes with measurable plastic-degrading activity, though none performed as well as an established plastic-eating enzyme. This work advances the search for biological tools that could help break down microplastic pollution in the environment.
Discovery and Biochemical Characterization of a Novel Polyesterase for the Degradation of Synthetic Plastics
Researchers used bioinformatics to discover a new enzyme from soil bacteria capable of breaking down synthetic plastics like PET and polyurethane. The enzyme was successfully expressed and characterized in the lab, offering a promising lead for developing biological plastic recycling approaches.
A high‐throughput expression and screening platform for applications‐driven PETase engineering
Researchers developed a high-throughput platform for engineering PETase enzymes — which break down plastic polyester — by using secretory expression to eliminate purification steps, enabling faster screening of enzyme variants for industrial plastic biodegradation applications.
Characterization and engineering of a plastic-degrading aromatic polyesterase
Researchers characterized and engineered an aromatic polyesterase enzyme capable of degrading plastic polymers, improving its activity through protein engineering and demonstrating its potential as a tool for biodegradation-based plastic cleanup.
Microbial enzymes for the recycling of recalcitrant petroleum‐based plastics: how far are we?
This review examines the progress in identifying microbial enzymes capable of breaking down petroleum-based plastics like polyethylene, polystyrene, polyurethane, and PET. Researchers highlight recent advances in using polyester-degrading enzymes to recover raw materials from PET waste through biocatalytic recycling. The study discusses the potential and remaining challenges of using biological approaches to address the growing global problem of plastic waste accumulation.