Biocatalysis in organic solvents (OSs) has found various important applications, particularly in organic synthesis and for the production of pharmaceuticals, flavors, and fragrances. However, the use of enzymes in OSs often results in enzyme deactivation or a dramatic drop in catalytic activity. Herein, we have developed a comprehensive understanding of the interactions between enzymes and OSs based on numerous observables obtained from molecular dynamics simulation of 32 variants of Bacillus subtilis lipase A (BSLA). We have tested the wild-type enzymes and variants carrying single and multiple substitutions toward the organic cosolvent 2,2,2-trifluoroethanol (TFE, 12% (v/v)). After analyzing the distribution of 35 structural and dynamic observables, we uncovered that increased enzyme surface hydration of substituted sites is the predominant factor to drive the improved resistance in OS. The iterative recombination of four surface substitutions revealed that the extent of hydration in BSLA variants correlates strongly with its OS resistance (R 2 = 0.91). Remarkably, the substitutions recombination led to a highly resistant BSLA variant (I12R/M137H/N166E) with a 7.8-fold improved resistance in 12% (v/v) TFE, while retaining comparable catalytic activity (∼92%) compared to the wild-type enzyme. Our findings prove that strengthening protein surface hydration via surface charge engineering is an effective and efficient rational strategy for tailoring enzyme stability in OSs.
Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However,n ative enzymes often have lowa ctivity and resistance in OSs and at elevated temperatures.H erein, we report as mart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme,B acillus subtilits Lipase A( BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems.I terative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance towardt hree OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and halflife up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 8 8C. The salt bridge redesign provides protein engineers with ap owerful and likely general approach to design OSsand/or thermal-resistant lipases and other a/b-hydrolases.
Escherichia coliscopper efflux oxidase (CueO) has rarely been employed in the cathodic compartment of enzymatic biofuel cells (EBFCs) due to its low redox potential (0.36 Vv s. Ag/AgCl, pH 5.5) towards O 2 reduction. Herein, directed evolution of CueO towards am ore positive onset potential was performed in an electrochemical screening system. An improved CueO variant (D439T/L502K) was obtained with asignificantly increased onset potential (0.54 V), comparable to that of high-redox-potential fungal laccases. Upon coupling with an anodic compartment, the EBFC exhibited an open-circuit voltage (V oc )o f0 .56 V. Directed enzyme evolution by tailoring enzymes to application conditions in EBFCs has been validated and might, in combination with molecular understanding,e nable future breakthroughs in EBFC performance
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