How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

Abstract

Biocatalysis in ionic liquids (ILs) gained substantial interest due to solvent properties of the ILs, such as near-zero vapor pressure, high thermal stability, and wide tunability. Enzymes are often not catalytically active in ILs; therefore, understanding and improving enzyme resistance in ILs are essential to enable efficient biocatalysis in ionic liquids. Surface charge engineering has repeatedly been reported to enhance enzyme resistance toward ILs. However, the molecular knowledge about how substitutions to charged amino acids improve enzyme activity in an IL is far from being understood. Here, we report a comprehensive study to provide some principles of how surface charged amino acid substitutions (negatively and positively) strengthen the IL resistance of the Bacillus subtilis lipase A (BSLA) in [BMIM]Cl. Twenty typical BSLA substitutions (ten beneficial and ten nonbeneficial, obtained from the BSLA-SSM library) were studied by molecular dynamics (MD) simulations in the [BMIM]Cl system. The BSLA-IL interaction patterns were printed by analyzing several structural-and solvation-based observables. Lessons learned by analyzing the SSM library of BSLA comprise the following: (i) A general trend was found where both negatively and positively charged substitutions increased the essential water molecules locally at the substitution site, thereby contributing to the overall protein hydration shell. (ii) Electrostatic repulsion of both IL ions and the refined hydration shell are ultimately the two main drivers to enhanced IL resistance. The analysis of 20 BSLA substitutions and the identified common interactions reveals that surface charge engineering is very likely to be a general protein engineering strategy to enhance lipase/enzyme activity in ILs. Moreover, this study also suggests that MD is a valuable technique to screen for beneficial substitutions that repel/recruit surface solvation.

Enzyme Hydration: How to Retain Resistance in Ionic Liquids

“…The BMIM + cation topology was obtained using the ATB online server with the GROMOS96 54a7 force field based on previous reports. ,,, Due to the PDB2PQR server’s speed and accuracy compared to other methods, protonation states were assigned based on predicted p K a utilizing the PROPKA technique at pH 7.4 . The protonation state of catalytic residues such as H156 was assigned with respect to previous studies using pdb2gmx. ,, The simulation parameters such as box size (cubic, 10 Å from the box edge), SPC/E water model, , system ionization, , neutralization, energy minimization, and NVT and NPT ensembles follow our previous study with a time step of 2 fs . The 100 ns production run was carried out with three independent runs using the NPT ensemble (2 fs time step at 300 K) as previously reported .…”

“…The main conclusion revealed that up to 13% of substitutions have improved resistance, providing a desirable source for fully understanding the protein–IL interaction. Meanwhile, the BSLA–SSM library holds more promise of unmasking the general design principle(s) than the traditional directed evolution campaign, as relatively few amino acid substitutions have been studied in comparison to the diversity that nature provides. , In our prior work, we successfully used atomistic MD simulation to explore dynamic interactions of ILs and enzyme variants at site resolution. ,, However, a large-scale statistical exploration based on more variants in multiple ILs is required to print a comprehensive landscape.…”

Molecular simulations explain the exceptional thermal stability, solvent tolerance and solubility of protein–polymer surfactant bioconjugates in ionic liquids

“…77,87 Further, MD simulations have shown surface charge and surface polar engineering to be successful strategies to enhance the IL/ polar solvent tolerance of a lipase. 88,89 Pramanik et al also demonstrated that the reduction in the interaction between the IL cation and the LipA surface is an efficient approach to stabilize LipA in a binary mixture of IL and water. 83,88 In the PS-IL forms, the surfactant layer in SFPLs imparts IL tolerance to the protein by lowering the concentration of both the cation and anion of IL in the vicinity of the protein surface, i.e., by screening the protein-IL interactions (refer to Fig.…”

“…88,89 Pramanik et al also demonstrated that the reduction in the interaction between the IL cation and the LipA surface is an efficient approach to stabilize LipA in a binary mixture of IL and water. 83,88 In the PS-IL forms, the surfactant layer in SFPLs imparts IL tolerance to the protein by lowering the concentration of both the cation and anion of IL in the vicinity of the protein surface, i.e., by screening the protein-IL interactions (refer to Fig. 4 and Table S5, ESI †).…”

“…4). The surfactant layer plays a similar role as that of the surface engineering strategy 77,83,87,88 to improve the IL tolerance of proteins. The ratio of the number of cations to anions present in the immediate vicinity of a protein in the P-IL systems, and hence the dominant destabilizing ion of the IL, is dependent on the nature of the protein surface (refer to Table S5, ESI †).…”