Functional display of active tetrameric beta-galactosidase using Bacillus subtilis spore display system

Area of Science:

• Microbiology
• Biotechnology
• Enzyme Engineering

Background:

• Functional display of active multimeric enzymes on bacterial surfaces is challenging due to assembly requirements.
• Existing surface display systems often fail to maintain the quaternary structure of multimeric proteins.
• Bacillus subtilis spores offer a robust platform for displaying proteins due to their stability.

Purpose of the Study:

• To develop a novel surface display system for functional bacterial display of active multimeric enzymes.
• To achieve the display of active tetrameric beta-galactosidase on Bacillus subtilis spores.
• To evaluate the impact of spore surface display on enzyme activity and spore integrity.

Main Methods:

• Engineered Bacillus subtilis spores using cotE and cotG genes as anchoring motifs.
• Fused beta-galactosidase (LacZ) gene with anchoring motifs for surface display.
• Verified surface localization using Miller assay, protease accessibility, and flow cytometry.
• Assessed spore wall integrity via lysozyme and heat treatments.
• Evaluated heat stability of the displayed enzyme.

Main Results:

• Successfully displayed functional tetrameric beta-galactosidase on the surface of Bacillus subtilis spores.
• CotG-LacZ fusion maintained spore wall integrity, while CotE-LacZ fusion showed some disruption.
• Surface localization of displayed beta-galactosidase was confirmed through multiple analytical methods.
• The displayed enzyme exhibited similar heat stability to the free enzyme.

Conclusions:

• The developed Bacillus subtilis spore-based system enables the functional display of active multimeric enzymes.
• CotG is a suitable anchoring motif for displaying beta-galactosidase on spores without compromising spore integrity.
• This platform holds promise for applications requiring stable and active enzyme presentation on bacterial spores.