Engineering subtilisin E for enhanced stability and activity in polar organic solvents

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Kinetics

Background:

• Subtilisin E is a widely studied serine protease.
• Aqualysin I is a thermophilic subtilisin-type protease providing structural insights.
• Protein engineering can enhance enzyme properties like stability and activity.

Purpose of the Study:

• To engineer a novel disulfide bond in subtilisin E to improve its properties.
• To investigate the role of electrostatic interactions in enzyme catalysis and stability.
• To enhance the stability and catalytic activity of subtilisin E in organic solvents.

Main Methods:

• Site-directed mutagenesis was used to introduce cysteine substitutions.
• Disulfide bond formation was confirmed in Bacillus subtilis.
• Enzyme activity and stability were measured using authentic peptide substrates and varying solvent conditions.

Main Results:

• A Cys170/Cys195 disulfide mutant showed a 5-10 fold increase in specific activity but a 50% decrease in catalytic efficiency.
• The disulfide mutant was thermolabile but stabilized relative to its reduced form.
• Disulfide mutants exhibited enhanced stability in polar organic solvents like dimethylformamide and ethanol.
• A quintet mutant with an additional amino-terminal disulfide bond and Ile31Leu mutation showed a 3-4 fold increase in catalytic efficiency in dimethylformamide.

Conclusions:

• Electrostatic interactions between Lys170 and Glu195 are crucial for subtilisin E catalysis and stability.
• Engineered disulfide bonds can enhance enzyme stability in organic solvents.
• Further protein engineering can optimize subtilisin E for applications in organic media.