IMMOBILIZATION OF LEAF BRANCH COMPOST CUTINASE (LCC) ONTO BACILLUS SUBTILIS SPORES FOR EFFICIENT PLASTIC DEGRADATION

Enzymes provide an efficient, clean, and reliable means of performing specific chemical reactions and have found use in the agricultural, food, chemical, and pharmaceutical industries. Despite the numerous advantages of using enzymes, their application is hindered by a lack of stability in harsh conditions, cost of production, and purification. A potential strategy for mitigating these shortcomings is the anchoring of enzymes onto bacterial spores. Spores are structures produced by certain bacteria to help them survive extreme environmental conditions. Fusing enzymes to the surface of spores via spore display systems can increase their thermal and pH stability while substantially simplifying the enzyme purification process. In this work, we present the construction of a Bacillus subtilis spore display system for the immobilization of the benchmark plastic-degrading enzyme LCCICCG using the spore coat protein CotG. We established the optimal sporulation conditions for spore yield in terms of incubation temperature and time. The stability of spores and the immobilized enzyme was tested at high temperatures, high salt concentrations and in organic solvents. Additionally, we tested the esterase activity of spores produced at different temperatures and determined that spores produced at 25°C for 24 h had the highest activity. The most active spores were used in poly(ethylene terephthalate) (PET) and poly(caprolactone) (PCL) degradation experiments, proving the spores could be used as efficient biocatalysts for plastic degradation. In addition to the previously mentioned benefits, the use of spores as plastic-degrading biocatalysts opens up opportunities for coupling the degradation process with biosynthetic processes for the up-cycling of released monomers. This innovative approach paves the way for sustainable plastic waste management and biotechnological advancements.