Enhancing the thermal stability of L-threonine dehydrogenase through computational-driven combinatorial mutagenesis

Area of Science:

• Enzyme Engineering
• Protein Engineering
• Industrial Biotechnology

Background:

• L-threonine dehydrogenases (L-TDHs) are vital industrial enzymes for producing L-2-aminoethyl acetate.
• The L-threonine dehydrogenase from E. coli (EcTDH) has high catalytic efficiency but poor thermal stability, limiting its industrial use.

Purpose of the Study:

• To enhance the thermal stability of EcTDH for high-temperature industrial applications.
• To investigate the effects of semi-rational design on EcTDH stability and activity.

Main Methods:

• Employed a semi-rational design strategy to create a combinatorial mutant (N221W/T248L) of EcTDH.
• Evaluated the thermal stability and catalytic properties of the engineered mutant compared to the wild-type enzyme.

Main Results:

• The N221W/T248L mutant demonstrated significantly improved thermal stability, with a half-life at 50°C (115 min) that is 3.2 times longer than the wild-type (35 min).
• The mutant retained comparable specific activity (12.01 U mg⁻¹) and catalytic efficiency (4.25 s⁻¹ mM⁻¹) to the wild-type enzyme.
• The enhanced stability makes EcTDH more suitable for high-temperature industrial processes.

Conclusions:

• Semi-rational protein engineering can effectively improve the thermal stability of industrial enzymes like EcTDH.
• The N221W/T248L mutant offers a promising alternative for industrial applications requiring thermostable L-TDHs.
• This strategy serves as a valuable reference for optimizing other industrial enzymes through directed evolution.