Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression

Area of Science:

• Biocatalysis and Enzyme Engineering
• Molecular Biology
• Protein Engineering

Background:

• Subtilisin E is a serine protease with native amidase activity.
• Expanding enzyme activity to ester substrates is crucial for biocatalysis.
• Directed evolution offers a powerful strategy for enzyme functional diversification.

Purpose of the Study:

• To engineer subtilisin E for enhanced esterase activity on diverse substrates.
• To develop a general esterolytic enzyme from a native amidase.
• To explore the application of molecular evolution in creating novel biocatalysts.

Main Methods:

• Functional periplasmic enzyme expression in Escherichia coli.
• Random mutagenesis and DNA shuffling for library generation.
• Cell-based kinetic screening using ester and amide substrates (Phe-NPE, S1'A, and tetrapeptide amide).

Main Results:

• Directed evolution successfully enhanced subtilisin E's activity on two distinct ester substrates, N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A).
• Three mutant enzymes demonstrated increased esterolytic activity on both Phe-NPE and S1'A.
• The evolved mutants retained similar amidase activity to the parental enzyme, indicating a broadened substrate specificity.

Conclusions:

• A general esterolytic subtilisin was successfully evolved using a combination of molecular evolution techniques.
• The engineered enzyme exhibits improved catalytic efficiency on structurally distinct ester substrates.
• This work highlights the potential of enzyme evolution for developing versatile biocatalysts for chemical synthesis.