High-Throughput Screening for Thermostable Polyester Hydrolases.

ABSTRACT

Due to the promise of more sustainable recycling of plastics through biocatalytic degradation, the search for and engineering of polyester hydrolases have become a thriving field of research. Furthermore, among other methods, halo formation assays have become popular for the detection of polyester-hydrolase activity. However, established halo-formation assays are limited in their ability to screen for thermostable enzymes, which are particularly important for efficient plastic degradation. The incubation of screening plates at temperatures above 50 °C leads to cell lysis and death. Therefore, equivalent master plates are commonly required to maintain and identify the active strains found on the screening plates. This replica plating procedure necessitates 20- to 60-fold more plates than our method, assuming the screened library is transferred to 384-well microtiter plates or 96-well microtiter plates, respectively, to organize the colonies in a retraceable manner, thus significantly lowering throughput. Here, we describe a halo formation assay that is designed to screen thermostable polyesterases independent of master plates and colony replication, thereby markedly reducing the workload and increasing the throughput.