Rational Tunnel Engineering for Enhanced Stability of Lipase in Organic Solvents

Lipases are well-studied robust enzymes for biocatalysis in organic solvents. Under non-aqueous conditions, they perform reactions such as esterification and transesterification. Despite great progress in enzyme engineering, improved stability in denaturing solvents is still warranted. Organic solvents have been reported to influence the hydration state of the enzyme, impart local thermodynamic changes, cause conformational fluctuations, all leading to decrease and even loss in activity.

Our group has engineered a lipase from the thermophilic bacterium Geobacillus stearothermophilus T6 (LipT6) towards higher stability in methanol using random mutagenesis and structure-guided consensus approaches and immobilized it in a sol-gel matrix enabling the reuse in successive reaction cycles. A rational approach was applied here using the recently solved crystal structure whereby solvent tunnels were altered to increase stability. Tunnel properties (diameter, length, hydrophobicity, etc.) can be modified to increase lipase organic solvent resistance by limiting diffusion of polar solvent molecules towards the enzyme`s inner core. MOLE 2.0 online generator was used for detecting cavities and tunnels in LipT6 crystal structure (PDB: 4X6U). The analysis yielded 9 solvent tunnels and the closest residues to these channels (4-5Å) were identified. Rational design of mutations was performed to block these tunnels by incorporating bulky residues. Activity screening following incubation in 60% methanol yielded 4 single mutants with higher stability compared to WT, ranging between 5- to 80-fold. Most of the effective residues were in the vicinity of the active site. In addition, T_m determination using nanoDSF showed elevated stability of these mutants in methanol, ethanol, dimethylsulfoxide and acetonitrile. Combinations of the stabilizing mutations was applied along with crystallography efforts towards profounder understanding of the substitutions’ effect on LipT6 structure.