A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in...

Area of Science:

• Enzymology
• Biotechnology
• Protein Engineering

Background:

• Esterases are crucial enzymes in various industrial applications.
• The identification and characterization of novel esterases with unique substrate specificities are of significant interest.
• Bacillus subtilis is a well-known source of industrially relevant enzymes.

Purpose of the Study:

• To clone and functionally express an esterase from Bacillus subtilis DSM402 (BS2) in E. coli.
• To characterize the enzymatic properties, including optimal temperature, Vmax, and Km.
• To explore the potential applications of BS2 esterase in resolving tertiary alcohol acetates and deprotecting peptides.

Main Methods:

• Gene cloning and functional expression of Bacillus subtilis esterase (BS2) in E. coli.
• Enzyme activity assays using p-nitrophenyl acetate as substrate.
• Determination of kinetic parameters (Vmax and Km) and optimal temperature.
• Application of the enzyme for chiral resolution of tertiary alcohol acetates and deprotection of tert-butyl ester groups from peptides.

Main Results:

• The cloned esterase (BS2) was successfully expressed in E. coli.
• BS2 exhibited optimal activity up to 50°C, with Vmax of 1449 mM/min and Km of 119 mM.
• The enzyme demonstrated the ability to resolve racemic acetates of tertiary alcohols.
• BS2 effectively removed tert-butyl ester protecting groups from peptides and showed promiscuous amidase activity.

Conclusions:

• The Bacillus subtilis esterase (BS2) is a robust enzyme with potential for biotechnological applications.
• Its unique ability to act on tertiary alcohols makes it valuable for chiral resolutions and peptide synthesis.
• Further investigation into its promiscuous amidase activity could reveal additional applications.