Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

Area of Science:

• Enzymology
• Protein Engineering
• Microbial Biotechnology

Background:

• Subtilisin E, an alkaline serine protease from Bacillus subtilis, plays a vital role in various industrial applications.
• The catalytic triad and surrounding residues significantly influence protease activity and stability.
• Understanding residue-specific effects is key to protein engineering for enhanced function.

Purpose of the Study:

• To investigate the impact of amino acid substitutions at position 31 on subtilisin E activity.
• To identify specific residues that enhance the catalytic efficiency of subtilisin E.
• To explore the structure-activity relationship concerning residue 31 and protease function.

Main Methods:

• Site-directed mutagenesis was employed to introduce eight uncharged amino acids at position 31.
• Mutant subtilisin E enzymes were expressed in Escherichia coli and purified.
• Enzyme activity, substrate specificity, heat stability, and optimal temperature were analyzed for wild-type and mutant enzymes.

Main Results:

• Mutations at position 31 showed varied effects on enzyme activity, with Val and Leu substitutions yielding active enzymes.
• The Leu31 mutant exhibited a substantial increase in activity compared to the wild-type enzyme.
• The Leu31 replacement resulted in a 2-6 fold increase in catalytic efficiency (kcat/Km) for peptide substrates, primarily due to an elevated kcat.

Conclusions:

• A branched-chain amino acid at position 31 is essential for optimal subtilisin E activity.
• The specific side chain structure of the amino acid at position 31 dictates the level of enzyme activity.
• Engineering subtilisin E at position 31 offers a viable strategy for developing highly active protease variants for biotechnological applications.