Wang_2023_Angew.Chem.Int.Ed.Engl__e202315125

Deep eutectic solvents (DESs), heralded for their synthesis simplicity, economic viability, and reduced volatility and flammability, have found increasing application in biocatalysis. However, challenges persist due to a frequent diminution in enzyme activity and stability. Herein, we developed a general protein engineering strategy, termed corner engineering, to acquire DES-resistant and thermostable enzymes via precisely tailoring of the transition region in enzyme structure. Employing Bacillus subtilis lipase A (BSLA) as a model, we delineated the engineering process, yielding five multi-DESs resistant variants with highly improved thermostability, such as K889E/N89K exhibited up to a 10.0-fold catalytic efficiency (kcat/KM) increase in 30% (v/v) ChCl:acetamide and 4.1-fold in 95% (v/v) ChCl:ethylene glycol accompanying 6.7-fold thermal resistance improvement than wild type at ~50 degreesC. The generality of the optimized approach was validated by two extra industrial enzymes, endo-beta-1,4-glucanase PvCel5A (used for biofuel production) and esterase Bs2Est (plastics degradation). The molecular investigations revealed that increased water molecules at substrate binding sites and finetuned helix formation at the corner region are two dominant determinants governing elevated resistance and thermostability. This study, coupling corner engineering with obtained molecular insights, illuminates enzyme-DES interaction patterns and fosters the rational design of more DES-resistant and thermostable enzymes in biocatalysis and biotransformation.