Enabling biocatalysis in high‐concentration organic cosolvent by enzyme gate engineering
Feng Cheng, Ming‐You Li, Dian‐Ju Wei, Xiao‐Jian Zhang, Dongxu Jia, Zhi‐Qiang Liu, Yu‐Guo Zheng
Abstract
Abstract Biocatalysis in high‐concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R ‐selective ω‐amine transaminase variant ( Ac ATA M2 ) exhibited high activity toward 50 g/L pro‐sitagliptin ketone 1‐[1‐piperidinyl]‐4‐[2,4,5‐trifluorophenyl]‐1,3‐butanedione (PTfpB). However, Ac ATA M2 displayed unsatisfactory organic–cosolvent resistance against high‐concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate‐binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of Ac ATA M2 , we identified the beneficial residues at the “enzyme gate” region via computational analysis, alanine scanning, and site‐saturation mutagenesis. Two beneficial variants, namely, Ac ATA M2 F56D and Ac ATA M2 F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half‐life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate‐binding pocket. Moreover, in the kilogram‐scale experiment, the conversion of 80 g/L substrate was increased from 50% ( Ac ATA M2 ) to 85% (M2 F56D in 40% DMSO) with a high e.e . of >99% within 24 h.