Rational Molecular Design for Improved ZHD101 Thermal Stability Based on the Introduction of Disulfide Bonds at the...

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Chemistry

Background:

• Zearalenone is a harmful mycotoxin in animal feed.
• Zearalenone hydrolase (ZHD101) degrades zearalenone but has poor thermal stability, limiting its industrial use.

Purpose of the Study:

• To enhance the thermal stability of Zearalenone hydrolase (ZHD101) through rational molecular design.
• To develop an effective strategy for improving protein thermal stability for industrial applications.

Main Methods:

• Rational molecular design involving disulfide bond introduction for dimerization.
• Analysis of atomic position fluctuations and dynamic information to identify high-vibration hotspots.
• Virtual saturation mutagenesis and conformational free energy calculations to create precise mutants.

Main Results:

• A ZHD101 variant (ZHD101^T229C/D170C) showed significantly improved thermal stability.
• The variant's half-inactivation temperature (T₅₀) increased by 7 °C, and its half-life (t_1/2) at 50 °C doubled.
• Melting temperature (T_m) increased by 18.1 °C, the highest reported for Zearalenone hydrolases.

Conclusions:

• A combination mutation strategy involving disulfide bonds and B-factor analysis effectively enhanced ZHD101 thermal stability.
• The engineered ZHD101^T229C/D170C variant is suitable for industrial applications.
• The developed strategy serves as a valuable reference for modifying and optimizing the thermal stability of other proteins.