Tailoring <i>Candida antarctica</i> Lipase B with Enhanced Transesterification Activity and Its Immobilization on Magnetic Nanocarbon-MgFe ₂ O ₄ Particles Enabling Sustainable Biodiesel Preparation

Engineering robust and recyclable biocatalysts is the foundation of sustainable biodiesel for renewable energy. Herein, a semirational design strategy was employed to tailor the catalytic pocket of <i>Candida antarctica</i> lipase B (CalB) targeting to enhance the catalytic performance. With assistance of AlphaFold3 modeling, the best multisite variant (MS-1) with mutants of L144V, I285P, and V286M in tailored catalytic pocket was obtained, showing a 10% increase in biodiesel conversion rate from 79% to 89% and a 1.44 times increase in transesterification activity over wild-type CalB from 28.11 to 40.67 U/mg. It was observed that the closer distance between the carboxyl carbon from the active site Ser¹⁰⁵ and the oxyanion hole Thr⁴⁰ in the CalB-triolein complex facilitated the greater transesterification activity. Meanwhile, nanocarbon-based magnetic particles (nanocarbon-MgFe₂O₄) were prepared via co-precipitation by mixing ferric and magnesium ions and using cheap and readily available ink nanocarbon as cores, showing superparamagnetism property (45.5 emu/g) and 15-25 nm in diameter. The MS-1 variant was covalently immobilized on nanocarbon-MgFe₂O₄ particles displaying larger interface and higher enzyme-loading capacity. This robust biocatalyst retained 80.2% of its initial activity after 12 reuse cycles, showing excellent structural stability and reusability. Its facile magnetic separation and stably operable interface architecture significantly reduce catalyst costs and downstream processing burden, endowing it with great potential for industrial continuous-flow production of green and sustainable biodiesel.