Directed Evolution of a Thermostable Lipase for Enantioselective Synthesis of Chiral Pharmaceutical Intermediates in Continuous Flow Bioreactors

Enantioselective biocatalysis in continuous flow represents a transformative approach for the sustainable synthesis of optically pure pharmaceutical intermediates. However, the operational deployment of wild-type enzymes is frequently hindered by inadequate stereoselectivity, thermal instability, and rapid deactivation in organic co-solvents. In this study, we engineered the thermostable lipase from Geobacillus thermocatenulatus (BTL2) via combinatorial active-site saturation test (CASTing) and directed evolution to enhance its enantioselectivity toward bulky chiral secondary alcohols. A triple-mutant variant, BTL2-M3 (L214F/I258V/M317L), exhibited a dramatic 28-fold increase in enantioselectivity (enantiomeric ratio, E > 240) toward racemic 1-phenylethanol during transesterification compared to the wild-type enzyme. The engineered biocatalyst was covalently immobilized onto glutaraldehyde-functionalized magnetic mesoporous silica nanoparticles, achieving an enzyme loading of 78.4 mg/g support and maintaining >85% of its initial catalytic activity across 120 hours of continuous operation. Integrated into a packed-bed microfluidic continuous flow reactor, the immobilized BTL2-M3 system achieved near-theoretical conversion (49.6%) and exceptional enantiomeric excess (>99.2% ee_p) at a residence time of only 4.2 minutes at 55 °C in toluene. The space-time yield reached 1.84 kg L⁻¹ h⁻¹, representing a 14-fold improvement over traditional batch reactors. This integrated protein engineering and flow biocatalysis strategy establishes a robust platform for the continuous, stereoselective production of high-value chiral pharmaceutical synthons.