2026-10-13T09:26:19Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34051401 2026-10-02T02:58:37Z category_1 category_4 category_8 category_21 category_873 category_46 category_734 portal_87 item_type_3 month_year_10_2026

Coated bacterial enzymes: a one-step approach for enzymatic purification and immobilization Inés Harguindeguy (25160565) Melisa S. Homse (25160568) Andrea C. Ramírez (25160571) Alan E. Sabetta (25160574) Sebastián F. Cavalitto (25160577) Gastón E. Ortiz (25160580) Biophysics Biochemistry Microbiology Biotechnology Chemical Sciences not elsewhere classified Immunology Biological Sciences not elsewhere classified β-Galactosidase Bifidobacterium bifidum coated bacterial enzymes enzyme immobilization enzyme purification SlpA domain <p>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 <i>Bifidobacterium bifidum</i> of dairy relevance. The chimeric enzyme BbgII‑SlpA was expressed in <i>Escherichia coli</i> and captured from crude lysate onto glutaraldehyde‑inactivated <i>Bacillus subtilis</i> cells via SlpA domain. Binding was characterized by a dissociation constant (K_d) of 16.2 μM and maximum binding capacity (B_max) of 144 μmol/g. The resulting CBEs 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 CBEs retained >80% activity after 390 min at 45 °C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 × 10⁶ M⁻¹·s⁻¹ for ONPG and 4.40 × 10² M⁻¹·s⁻¹ for lactose. Moreover, CBEs showed improved tolerance to cations such as Ca²⁺ and Fe²⁺. Furthermore, at 7 °C in skim milk, the CBEs achieved approximately 53% lactose hydrolysis within 20 h and retained 35–45% of its initial activity after five reuse cycles.</p> 2026-10-02T02:58:37Z Dataset Dataset 10.6084/m9.figshare.34051401.v1 https://figshare.com/articles/dataset/Coated_bacterial_enzymes_a_one-step_approach_for_enzymatic_purification_and_immobilization/34051401 CC BY 4.0