A trehalase from Zunongwangia sp.: characterization and improving catalytic efficiency by directed evolution

Area of Science:

• Biochemistry
• Enzyme Engineering

Background:

• Trehalases are enzymes with diverse applications in food, insecticides, and plant biotechnology.
• A novel trehalase was identified from the marine bacterium Zunongwangia sp.

Purpose of the Study:

• To clone, characterize, and engineer a trehalase (TreZ) from Zunongwangia sp.
• To improve the enzyme's catalytic efficiency through directed evolution.

Main Methods:

• Gene cloning and sequencing of treZ.
• Enzyme activity assays under varying temperature, pH, and NaCl concentrations.
• Error-prone PCR and high-throughput screening for enzyme engineering.
• Site-directed mutagenesis to analyze specific mutations.
• Structural modeling and substrate docking.

Main Results:

• A novel α,α-trehalase (TreZ) was cloned and characterized, with optimal activity at 50°C and pH 6.
• Enzyme activity was enhanced by NaCl, showing 136% activity at 1M.
• Directed evolution produced variant C4 with a 3.3-fold higher catalytic efficiency (kcat/Km) due to mutations Y227H and R442G.
• Both Y227H and R442G mutations significantly contributed to the enhanced catalytic efficiency.

Conclusions:

• A novel, engineered trehalase (TreZ) with significantly improved catalytic activity was developed.
• Key mutations (Y227H, R442G) were identified and validated for their role in enhancing enzyme efficiency.
• Structural insights suggest mutations improve substrate binding and product release mechanisms.