Immobilization of alginate C-5 epimerases using Bacillus subtilis spore display

Alginates are the most abundant polysaccharides found in brown seaweed, composed of (1 -> 4)-linked beta-D-mannuronate (M) and its C-5 epimer, alpha-L-guluronate (G). The G-blocks of alginate possess viscosifying and gelling properties, making alginates valuable industrial polysaccharides. Alginate epimerases are enzymes epimerizing M to G, enhancing the usability and value of alginate. The three alginate epimerases AlgE1, AlgE4, and AlgE6 were immobilized using Bacillus subtilis spores displaying the epimerases fused to the spore crust protein CotY. To our knowledge, this is the first display of immobilized alginate-modifying enzymes. Activity assays of the four AlgE4-displaying spore strains showed that AlgE4 produced MG-blocks from polyM alginate. AlgE4 was tested linked by its N- and C-termini. Two linkers with different flexibility were tested, both containing a TEV protease cleavage site. Immobilizing alginate epimerases on B. subtilis spores resulted in a recyclable system that is easy to isolate and reuse, thus opening possibilities for industrial application. Recyclability was demonstrated by performing five consecutive reactions with the same batch of AlgE4 spores, with the spores retaining 24% of the starting activity after four rounds of reuse. TEV cleavage of spore-displayed enzyme was optimized using spores displaying a green fluorescent protein, and these optimized conditions were used to cleave AlgE4 off the spores. The cleavage of four AlgE4-displaying spores was successful, but cleavage efficiency varied depending on which terminus of AlgE4 was fused to CotY. IMPORTANCE Seaweed is a scalable resource that requires no fresh water, fertilizer, or arable land, making it an important biomass for bioeconomies. Alginates are a major component of brown seaweed and are widely used in food, feed, technical, and pharmacological industries. To tailor the functional properties of alginates, alginate epimerases have shown to be promising for postharvest valorization of alginate. This study investigates an efficient and easy method to produce immobilized alginate epimerases, thus opening new industrial use cases. In this study, the alginate epimerases are immobilized on the surface of Bacillus subtilis spores. The bacterium forms spores in reaction to nutrient starvation, which are highly resistant to external influences and can be repurposed as a stable protein display platform for numerous applications due to its ease of genomic manipulation and cultivation.