School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing, 210097, China.
RWTH Aachen University, Templergraben 55, 52062, Aachen, Germany.
Angew Chem Int Ed Engl. 2024 Jan 8;63(2):e202315125. doi: 10.1002/anie.202315125. Epub 2023 Dec 6.
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 (k /K ) 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.
深共晶溶剂（DESs）以其合成简单、经济可行、挥发性和可燃性降低而备受关注，在生物催化中得到了越来越多的应用。然而，由于酶活性和稳定性经常降低，仍然存在挑战。在此，我们开发了一种通用的蛋白质工程策略，称为角工程，通过精确调整酶结构的过渡区来获得 DES 抗性和热稳定的酶。我们以枯草芽孢杆菌脂肪酶 A（BSLA）为模型，描述了工程化过程，得到了五个具有高度改善热稳定性的多 DES 抗性变体，例如 K88E/N89K，在 30%（v/v）氯化胆碱（ChCl）：乙酰胺和 95%（v/v）ChCl：乙二醇中的催化效率（k /K）分别提高了 10.0 倍和 4.1 倍，在 ≈50°C 时比野生型的耐热性提高了 6.7 倍。通过两种额外的工业酶——内切-β-1,4-葡聚糖酶 PvCel5A（用于生物燃料生产）和酯酶 Bs2Est（用于塑料降解）验证了优化方法的通用性。分子研究表明，增加底物结合裂隙中的水分子和微调角区的螺旋形成是提高抗性和热稳定性的两个主要决定因素。这项研究将角工程与获得的分子见解相结合，阐明了酶-DES 相互作用模式，并促进了在生物催化和生物转化中设计更多的 DES 抗性和热稳定酶的合理设计。
