Coated Bacterial Enzymes: A one-step approach for enzymatic purification and immobilization

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a β-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 µM and maximum binding capacity (Bmax) of 144 µmol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 °C for ONPG and 50 °C for lactose, and CBE retained >80% activity after 390 min at 45 °C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 ×10 ⁶ M ^-1 ·s ^-1 for ONPG and 4.40 ×10 ² M ^-1 ·s ^-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca ²⁺ and Fe ²⁺ . These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.