Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha

The ability to catalyze diverse reactions with relevance for chemical and pharmaceutical research and industry has led to an increasing interest in fungal enzymes. There is still an enormous potential considering the sheer amount of new enzymes from the huge diversity of fungi. Most of these fungal enzymes have not been characterized yet due to the lack of high throughput synthesis and analysis methods. This bottleneck could be overcome by means of cell-free protein synthesis. In this study, cell-free protein synthesis based on eukaryotic cell lysates was utilized to produce a functional glycoside hydrolase (GH78) from the soft-rot fungus Xylaria polymorpha (Ascomycota). The enzyme was successfully synthesized under different reaction conditions. We characterized its enzymatic activities and immobilized the protein via FLAG-Tag interaction. Alteration of several conditions including reaction temperature, template design and lysate supplementation had an influence on the activity of cell-free synthesized GH78. Consequently this led to a production of purified GH78 with a specific activity of 15.4 U mg− 1. The results of this study may be foundational for future high throughput fungal enzyme screenings, including substrate spectra analysis and mutant screenings. •Cell-free protein synthesis of active rhamnosidase GH78 from Xylaria polymorpha with straightforward kinetic evaluation.•Parameters such as synthesis temperature and template modifications influence activity of a cell-free produced GH78.•Substrate specificity of GH78 depends on the synthesis process. A different rhamnosidase activity was detected.