Simulation-guided enzyme discovery: A new microbial source of cellobiose 2-epimerase

Area of Science:

• Biotechnology
• Enzyme Engineering
• Computational Biology

Background:

• Cellobiose 2-epimerase (CE) is valuable for the dairy industry.
• Thermostable CEs are crucial for enhanced lactulose production.
• Novel screening methods are needed for identifying thermostable enzymes.

Purpose of the Study:

• To evaluate molecular dynamics (MD) simulations for screening thermostable enzymes.
• To identify novel CEs with improved thermostability and activity.
• To assess the potential of Dictyoglomus thermophilum CE (Dith-CE) for lactulose production.

Main Methods:

• MD simulations were performed on eleven uncharacterized CE sequences.
• Experimental characterization of the most promising CE candidates.
• Kinetic parameter determination (kcat, Km) for isomerization activity.

Main Results:

• MD simulations successfully filtered potential thermostable CEs.
• Dith-CE exhibited high thermostability and superior epimerization and isomerization activities.
• Optimal activity for Dith-CE was observed at 85°C and pH 7.0.
• Kinetic parameters for Dith-CE isomerization were determined (kcat = 3.98 ± 0.3 s⁻¹, Km = 235.2 ± 11.2 mM).

Conclusions:

• MD simulation is a feasible preliminary tool for thermostable enzyme screening.
• Dith-CE is a highly promising enzyme for industrial lactulose production.
• Further research into Dith-CE can optimize its application in the dairy sector.