Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system

Area of Science:

• Enzymology
• Protein Engineering
• Biotechnology

Background:

• Subtilisin proteases are widely used enzymes.
• Developing enzymes with enhanced activity at low temperatures is crucial for various industrial applications.
• Evolutionary engineering offers a powerful approach to enzyme modification.

Purpose of the Study:

• To engineer a cold-adapted subtilisin protease with improved catalytic efficiency at low temperatures.
• To identify the specific mutations responsible for cold adaptation and enhanced activity.
• To analyze the impact of mutations on substrate affinity and overall enzyme stability.

Main Methods:

• Evolutionary engineering using sequential in vitro random mutagenesis.
• Screening for improved protease activity at low temperatures.
• Kinetic analysis (kcat/Km, Km) to determine catalytic efficiency and substrate affinity.
• Circular dichroism spectroscopy to assess structural stability.

Main Results:

• A mutant subtilisin (m-63) exhibited 100% higher catalytic efficiency at 10°C compared to the wild type.
• Three key mutations (V72I, A92T, G131D) were identified as responsible for cold adaptation.
• Increased substrate affinity (decreased Km) was the primary driver of enhanced activity.
• A92T and G131D mutations compensated for the negative activity contribution of the V72I mutation.
• Reduced structural stability was observed at elevated temperatures (60°C) and low temperatures (10°C).

Conclusions:

• Evolutionary engineering successfully generated a cold-adapted subtilisin with significantly enhanced catalytic efficiency at 10°C.
• The enhanced activity is mainly attributed to improved substrate binding, driven by specific mutations.
• While activity is enhanced, the cold adaptation strategy may impact overall protein stability.