Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability

Area of Science:

• Biochemistry
• Enzyme Engineering
• Biotechnology

Background:

• Carbon capture and storage (CCS) technologies are crucial for mitigating climate change.
• Carbonic anhydrases (CAs) are enzymes that catalyze CO2 hydration, making them promising for CO2 fixation.
• Thermostable CAs are needed for efficient CO2 capture at elevated temperatures.

Purpose of the Study:

• To rationally engineer the alpha-carbonic anhydrase (NtCA) from *Nitratiruptor tergarcus* for increased thermostability and CO2 hydration activity.
• To identify specific mutations that enhance both enzyme stability and catalytic efficiency at high temperatures.

Main Methods:

• In silico analysis using DEEPDDG software to predict stabilizing mutations.
• Site-directed mutagenesis to introduce 13 specific mutations in NtCA.
• Expression and characterization of wild-type and mutant NtCA in *Escherichia coli*.
• Assessing enzyme activity, thermostability, and structural changes using molecular dynamics simulations.

Main Results:

• Mutations D168K (surface) and R210L (dimeric interface) significantly improved NtCA's activity and stability.
• Most engineered mutants exhibited enhanced thermostability.
• Mutants R210K and N88K_R210L showed up to an 11-fold increase in CO2 hydration activity.
• Molecular dynamics simulations revealed structural alterations responsible for improved enzyme performance.

Conclusions:

• Rational engineering can enhance the thermostability and CO2 hydration activity of alpha-carbonic anhydrases.
• Specific mutations, particularly D168K and R210L, are effective for improving NtCA performance.
• The findings provide valuable insights for the development of engineered CAs for industrial CO2 capture applications.