Improved Soluble Expression and Catalytic Activity of a Thermostable Esterase Using a High-Throughput Screening...

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Expression

Background:

• Thermostable esterase Aaeo1 exhibits low expression and inclusion body formation in E. coli.
• Efficient production of functional enzymes is crucial for industrial applications.

Purpose of the Study:

• To develop a high-throughput screening method for improving soluble expression and activity of Aaeo1.
• To identify Aaeo1 mutants with enhanced catalytic efficiency and protein production.

Main Methods:

• Established a split-green fluorescent protein (GFP) system for correlating fluorescence with soluble protein levels and esterase activity.
• Performed primary high-throughput screening using flow cytometry with a split-GFP reporter.
• Conducted secondary screening based on esterase activity to select improved mutants.

Main Results:

• The split-GFP system showed a linear correlation between fluorescence intensity, soluble protein expression, and esterase activity.
• Two mutants with significantly improved soluble expression (2-fold increase) and catalytic activity (2-fold higher kcat/Km) were identified.
• One mutant (I51V-E170D) demonstrated a 4.5-fold increase in enzyme activity compared to the parent enzyme.

Conclusions:

• The split-GFP system is effective for high-throughput screening of enzyme variants with improved soluble expression and activity.
• Directed evolution using this system successfully generated Aaeo1 mutants with enhanced biochemical properties.
• The identified mutations provide insights into structure-function relationships for thermostable esterases.