Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

Computational design of disulfide bonds was performed for lipase from Geobacillus stearothermophilus T6 (LipT6) for enhanced methanol stability and improved biodiesel production. Thirteen double mutants comprising new cysteine pairs were screened and evaluated for their stability in 70 % methanol. Superior stability was found with variant E251C/G332C (M13) having a 5.5-fold higher hydrolysis activity and enhanced unfolding temperature (T_m) of +7.9 °C in methanol compared with wild-type. Moreover, M13 converted nearly 80 % waste chicken oil to biodiesel, representing a 2.4-fold improvement relative to the WT. Structural studies using X-ray crystallography confirmed the existence of the engineered disulfide bonds shedding light on the link between the bond location and backbone architecture with its stabilization impact. Rational integration of disulfide bonds is suggested to be a feasible method to promote elevated stability in organic solvents for various industrial applications such as biodiesel synthesis.