nanoDSF as screening tool for enzyme libraries and biotechnology development.

Area of Science:

• Biocatalysis
• Protein Engineering
• Enzyme Technology

Background:

• Industrial enzyme applications require high stability under harsh conditions (e.g., high substrate/cosolvent concentrations, non-neutral pH, elevated temperatures).
• Thermal stability is crucial for enzyme purification and process efficiency, but current high-throughput screening (HTS) methods are often slow and require extensive protein purification.

Purpose of the Study:

• To evaluate nanoscale differential scanning fluorimetry (nanoDSF) as a rapid and reliable HTS tool for determining enzyme melting points.
• To demonstrate the utility of nanoDSF for discovering thermostable enzymes from metagenomic libraries and screening protein variants.

Main Methods:

• Utilized hypotonic extraction for high-quality protein samples from bacterial cells.
• Employed nanoDSF to measure melting temperatures (Tm) of native proteins, enabling hundreds of measurements per day.
• Screened a diverse aldolase library and variants from saturation mutagenesis using nanoDSF.
• Investigated enzyme tolerance to cosolvents and substrate-induced stabilization.

Main Results:

• NanoDSF accurately determined melting points of native proteins, facilitating rapid screening.
• Hypotonic extraction improved sample quality, enabling efficient Tm determination.
• Identified novel thermostable enzymes and characterized enzyme variants.
• Demonstrated a linear decrease in Tm with increasing cosolvent concentration for multiple enzymes and cosolvents.
• Observed substrate-induced stabilization of hexokinase by sugars.

Conclusions:

• NanoDSF is a valuable tool for HTS of enzyme thermostability and discovery of biocatalysts.
• The method accelerates enzyme engineering and characterization for industrial viability.
• NanoDSF screening under various reaction conditions (cosolvents, substrates) provides insights into enzyme stability.