Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by...

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Mesophilic alkaline serine proteases are widely used industrially.
• Their activity significantly decreases at low temperatures, limiting applications.
• Improving low-temperature performance is crucial for these enzymes.

Purpose of the Study:

• To enhance the cold activity of a Bacillus pumilus alkaline serine protease (DHAP).
• To investigate the impact of directed evolution on protease performance at low temperatures.
• To determine if enhanced cold activity can be achieved without compromising thermostability.

Main Methods:

• Directed evolution was used to generate variants of DHAP.
• Site-directed mutagenesis was employed to create combined variants.
• Proteolytic activity and thermostability of wild-type and variants were assessed.
• Kinetic analysis and structural modeling were performed.

Main Results:

• Seven DHAP variants showed increased proteolytic activity at 15°C.
• Thermostability was generally maintained across the variants.
• Combined variants exhibited further increases in specific activity and catalytic efficiency (kcat).
• Specific variants (P9S/K27Q, P9S/T162I) showed a 5-fold increase in activity at 15°C with no loss of thermostability.

Conclusions:

• Directed evolution and mutagenesis successfully enhanced DHAP's low-temperature hydrolytic efficiency.
• Enhanced cold activity does not necessitate a loss of thermostability in bacterial proteases.
• It is possible to engineer protease variants with both improved cold activity and retained thermostability.