Multiple active forms of a novel serine protease from Bacillus subtilis

Area of Science:

• Microbiology
• Molecular Biology
• Enzymology

Background:

• Bacillus subtilis is a well-studied bacterium used for industrial enzyme production.
• Serine proteases are crucial enzymes with diverse biological functions.
• Understanding novel enzyme structures and functions is key to biotechnological advancements.

Purpose of the Study:

• To clone and sequence the gene encoding a novel serine protease from Bacillus subtilis.
• To investigate the functional domains of the novel serine protease.
• To characterize the active forms of the enzyme expressed in B. subtilis.

Main Methods:

• Gene cloning and sequencing of the epr gene from Bacillus subtilis.
• Expression of the epr gene in B. subtilis using a plasmid vector.
• Analysis of expressed protein forms using molecular mass determination (e.g., SDS-PAGE).
• Site-directed mutagenesis (gene deletions) to assess functional domains.

Main Results:

• The epr gene was successfully cloned and sequenced, revealing a novel serine protease.
• Active enzyme forms with molecular masses ranging from 40 to 34 kDa were detected in the culture medium.
• Deletions in the C-terminal region of the epr gene abolished larger enzyme species but retained the 34 kDa active form.
• The C-terminal third of the protein was found to be dispensable for protease activity.

Conclusions:

• The epr gene encodes a serine protease homologous to subtilisin, with a C-terminal domain of unknown function.
• The observed size variation in active enzyme forms is likely due to C-terminal processing or degradation.
• The C-terminal domain does not appear to be essential for the catalytic activity of the serine protease.