Corner Engineering: Tailoring Enzymes for Enhanced Resistance and Thermostability in Deep Eutectic Solvents

Abstract

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 precise 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 K88E/N89 K exhibited up to a 10.0‐fold catalytic efficiency (kcat/KM) increase in 30 % (v/v) choline chloride (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 °C. The generality of the optimized approach was validated by two extra industrial enzymes, endo‐β‐1,4‐glucanase PvCel5A (used for biofuel production) and esterase Bs2Est (used for plastics degradation). The molecular investigations revealed that increased water molecules at substrate binding cleft 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.

Solvent Tolerance Improvement of Lipases Enhanced Their Applications: State of the Art

“…It was noted that the degree of BSLA hydration is positively correlated with its tolerance in organic solvents and that penetration of polar organic solvents accompanied by stripping of water molecules results in the destruction of the hydration layer on the lipase’s surface, a decrease in the degree of hydration, and a corresponding decrease in the stability of the lipases [ 113 ]. Similar conclusions have been achieved by Pramanik et al [ 114 ] in the IL system and Wang et al [ 115 ] in the DES system. They found that compared to nonbeneficial variants and the wild type, the BSLA variants with increased solvent tolerance had a significantly higher number of water molecules on their surface.…”

Eutectogels as Promising Materials in Biocatalysis

“…[8] The unique industrially valuable advantages of many DES, such as low cost, easy preparation, low volatility, and non-toxicity, make them particularly attractive as enzymatic reaction media, and it has become a trending topic nowadays. [9][10][11] Several review articles addressing biocatalysis in DES have been published for interested readers. [12][13][14][15][16][17] In this context, we see an unparalleled opportunity for pushing this field toward enzyme immobilization into DEShosting soft materials, called "eutectogels", benefiting from the well-known advantages of solid supports such as higher enzyme stability, broader biocatalyst applicability, recycling, and continuous operation possibilities.…”