Engineering access tunnel dynamics of Candida antarctica lipase B enhances esterification of multiple bulky substrates

BACKGROUND: Lipase-catalyzed esterification in organic and solvent-rich systems offers a sustainable route for producing high-value esters, but conversion is often limited by accumulation of water generated during esterification and the steric demands of bulky substrates. RESULTS: Here, molecular dynamics (MD)-guided access-tunnel engineering was combined with a copper-soap high-throughput colorimetric assay to improve Candida antarctica lipase B (CALB)-catalyzed esterification. The previously reported thermostable variant M1 (L147S/T244P/S250P/T256D/N292D) was used as template to obtain an improvement of both activity and thermostability. M4 (L147S/T244P/S250P/T256D/N292D/L140W/L144F/I285F/V286L) was the best-performing variant and showed more than 4.5-fold higher volume-normalized apparent activity than wild-type (WT) in bulky-substrate esterifications, including phytosteryl oleate, glucose laurate, sucrose laurate, and α-tocopheryl succinate synthesis. Because succinic-anhydride acylation does not generate stoichiometric water, α-tocopheryl succinate synthesis was used only as a bulky-substrate-access probe. Structural and MD analyses indicated that substitutions near Trp140/Phe144 increased lid-region flexibility and remodeled the tunnel microenvironment, consistent with a putative relay-style water-transport model involving active-site Ser105 and tunnel-entrance Trp140. Immobilized M4@AOMR further achieved 98.6% conversion in preparative sucrose stearate synthesis. CONCLUSION: These results show that coordinated regulation of tunnel dynamics, interactions with reaction-generated water, and immobilized-enzyme performance is a potential strategy for ester synthesis; however, the steric and water-transport contributions were not quantitatively separated.