Cell-free pipeline for discovery of thermotolerant xylanases and endo-1,4-β-glucanases

- Putative enzymes from gene databases are becoming accessible due to low cost of gene synthesis.
- Cell-free protein expression allows the expression of identified putative genes.
- Novel glucanase and xylanase assays are compatible with cell-free produced proteins.
- A streamlined pipeline integrating the steps described above has been established.
- Putative glucanases and xylanases were characterized using this pipeline.

The rapid expansion in the number of sequenced genomes and metagenomes provides an exceptional resource for mining of the enzymes with biotechnologically relevant properties. However, the majority of protein production and analysis methods are not sufficiently cost-efficient and scalable to experimentally verify the results of computational genomic mining. Here, we present a pipeline based on Leishmania tarentolae cell-free system that was used to characterize 30 putative thermostable endo-1,4-β-glucanases and xylanases identified in public genomic databases. In order to analyse the recombinant proteins without purification, novel high-throughput assays for glucanase and xylanase activities were developed. The assays rely on solubilisation of labelled particulate substrates performed in multiwell plates. Using this approach both acidophilic and thermophilic enzymes were identified. The developed approach enables rapid discovery of new biotechnologically useful enzymes.