How To Engineer Ionic Liquids Resistant Enzymes: Insights from

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Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

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How To Engineer Ionic Liquids Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study Subrata Pramanik,†,∥ Gaurao V. Dhoke,†,∥ Karl-Erich Jaeger,‡ Ulrich Schwaneberg,†,§ and Mehdi D. Davari*,† †

Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf and Research Center Jülich, Wilhelm Johnen Strasse, 52426 Jülich, Germany § DWI − Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52074 Aachen, Germany

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‡

S Supporting Information *

ABSTRACT: Ionic liquids (ILs) are widely recognized as highly attractive solvents for biocatalysis due to their high stabilities and diverse tunable chemical properties. A major challenge for biocatalysis in ILs is the reduction of enzyme activity. Thus, molecular understanding of enzyme−ILs interactions is crucial to design enzymes for improved ILs resistance. Herein, we studied interactions of Bacillus subtilis lipase A (BSLA) and four commonly used imidazolium-based ILs (1-butyl-3-methylimidazolium (BMIM+) cation with Cl−, Br−, I−, and TfO− anions) using molecular dynamics simulations. Our results show that ILs cosolvents do not alter the overall and local BSLA conformation. However, the ILs effects on the reduction of activity is attributed to dominant surface interactions of BMIM+ cations that strip off essential water molecules from the BSLA surface. Solvent spatial distribution function analysis revealed that BMIM+ has a high binding affinity toward the BSLA surface via hydrophobic or π−π interactions. Interestingly, the comparison of simulation results with experimental full site saturation mutagenesis BSLA libraries confirmed that most of the beneficial positions for resistance improvement are located at the BMIM+ binding regions. These key findings suggest that reducing BMIM+ binding through surface charge engineering might be a general protein engineering strategy to improve BSLA resistance in ILs and is most likely applicable to other lipases and α/β-hydrolases. KEYWORDS: Protein engineering, Directed evolution, Biocatalysis, Ionic liquids, Molecular dynamics simulations, Lipase A

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nase P450 BM-320 tend to deactivate/destabilize in ILs, which may be due to the interaction of ILs with enzymes. Our previous report showed that the residual activity of Bacillus subtilis lipase A (BSLA) was reduced up to 30%−40% in the commonly used BMIM+-based ILs, including [BMIM][Cl] (18.3% v/v), [BMIM][Br] (13.2% v/v), [BMIM][I] (10% v/ v), and [BMIM][TfO] (15% v/v).15 First principles of enzymes destabilization in ILs are not well understood and remain challenging for chemo- and biocatalysis in ILs. In general, enzyme activity and stability in ILs rely on several features, including ion−protein contacts, ion−water interactions, inhibitory potential of ions with substrate, protein hydration, and conformation of the active site geometry.19,21−24 Previous simulation studies have shown the interactions of ILs on enzyme stability based on either IL mixtures (e.g., 10−95 wt % of [EMIM][OAC], [EMIM][EtSO4])19 or “pure” ILs (e.g., [BMIM][NO3]).25 Recently,

INTRODUCTION Ionic liquids (ILs) are important solvents for chemo- and biocatalysis.1 ILs are inherited with the ionic nature of inorganic salts and the organic nature of organic solvents that may undergo large structural diversity.2 Their unique properties include good solvation ability, low melting temperature, good conductivity, a wide electrochemical window, thermal and electrochemical stabilities, nonvolatility, and nonflammability.3,4 Due their properties, IL are highly attractive solvents from chemo- and biocatalysis points of view.3,5−7 ILs are increasingly drawing attention as green alternatives over volatile organic solvents as reaction solvents.5,8,9 Recently, ILs-based solvent systems have been reported to be promising for the dissolutions of biomass, the fractionation of biomass, and the enzymatic depolymerization of pretreated biomass.10,11 Stability of enzymes in ILs is an essential prerequisite to explore the synthetic application potential of ILs in biocatalysis, for instance, performing biomass depolymerization in homogeneous solutions.4,12−14 Most enzymes such as lipase,15−17 cellulase,18 laccase,7 xylanase,19 and monooxyge© 2019 American Chemical Society

Received: February 5, 2019 Revised: May 17, 2019 Published: May 28, 2019 11293

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

Research Article

ACS Sustainable Chemistry & Engineering

Protein engineering is a powerful tool for enzyme stabilization in ILs, and the first case studies on enzymes with improved IL resistance have been reported and provide first clues to an in depth understanding of enzyme stabilization in ILs.15,24,36 Recent comprehensive studies on lipase BSLA, in which full site saturation mutagenesis libraries at each position of the BSLA enzymes was generated and covered all natural amino acid exchanges at each position, concluded that global design principles of enzymes cannot be found in random mutagenesis libraries.15 The comparison of epPCR (random mutagenesis) libraries showed that in directed evolution campaigns only a small fraction of the natural diversity is generated per enzyme position due to the bias of the random mutagenesis methods and organization of the genetic code.37,38 Here, we use the data from the site saturation mutagenesis library of BSLA (BSLA-SSM library) for a comparative analysis and perform molecular dynamics (MD) simulations of BSLA in four ILs cosolvents (ILs cosolvents are referred to as ILs throughout the text) to understand how the individual ions affect structure and stability of BSLA at the molecular level. First, we investigate the effect of ILs on structural stability, flexibility, and solvent accessibility. Second, we describe detailed solvation phenomena such as solvents distribution, solvents conformation on a BSLA surface, and quantitative analysis of solvent interactions on a BSLA surface. Next, we compare simulation results with beneficial variants obtained in the BSLA-SSM library15 to validate the computational findings. Finally, we discuss how the gained knowledge can be used to develop a general protein engineering principle to stabilize α/ β-hydrolases in ILs. Overall, the present study provides a molecular insight into the effect of ILs on BSLA from MD simulations in comparison with the BSLA-SSM library to derive a general protein engineering approach to improve the resistance of α/β-hydrolases in ILs.

Kim et al. showed a comparative effect of cations on the activity and stability of Candida antarctica Lipase B (CALB) using [EMIM][TfO], [HMIM][TfO], and [OMIM][TfO].16 Kim et al. attributed the latter findings to the presence of long hydrophobic tails that facilitate ion−protein interactions and thereby cause structural distortions and a decreased CALB activity in ILs.16 Another study demonstrated the effect of ions by using [C4MIM][Cl], [C2MIM][Cl], [C4MIM][DCA], and [C2MIM][DCA] on protein immunoregulatory 7 (IM7).26 It was reported that size and hydrophobicity of cations are important for the destabilization of IM7, and it was shown that the interaction of C4MIM+ was more prevalent on denaturation than C2MIM+. The effect of anions showed that weakly hydrated DCA− anions strongly bind to positively charged or polar residues, leading to the partial dehydration of the backbone groups, whereas Cl− is the most hydrated anion and should therefore be less available to interact with IM7.26 Likewise, Pfaendtner and co-workers observed that substitution of lysine with glutamate on an enzyme surface decreases interactions with the anions, whereas cation interactions increase with Candida rugosa lipase and Bos taurus αchymotrypsin in [BMIM][Cl] (20 wt %) or [EMIM][EtSO4] (20 wt %), respectively.27 Further studies by Kaar and Nordwald indicated that binding of the BMIM+ cations were decreased (7- and 3.5-fold for chymotrypsin from bovine and Candida rugosa lipase, respectively) with an increasing ratio of positive to negative surface charges.28 The same report showed that lowering the ratio of positive to negative surface charges leads to preferential exclusion of Cl− anions and increased enzyme stability.28 Recent NMR studies revealed [BMIM][Cl]-induced structural perturbations near the catalytic triad (S77, D133, and H156) of BSLA, indicating the importance of direct ion interactions with the catalytic triad.29 It was shown that substitution of G158E near the catalytic triad resulted in a 2.5-fold resistance improvement of BSLA in [BMIM][Cl] (50% v/v) due to the inhibition of the hydrophobic interactions of BMIM+ cations with the catalytic triad. Similarly, another substitution K44E also significantly enhanced the resistance of BSLA,29 and it was suggested that K44E might diminish the attraction of Cl− anions on the BSLA surface. Additionally, BSLA contains an oxyanion hole (I12, M78) that stabilizes the negatively charged reaction intermediates.30 In addition to the catalytic triad, stabilization of the oxyanion hole might be essential to improve the resistance of BSLA in ILs.31,32 More recent crystallographic33 and simulation studies34 described that hydrophobic and cation−π interactions of BMIM+ with surface residues might be driving forces for the unfolding and destabilization of lipase in ILs. A recent simulation study from our group demonstrated that the hydrogen bond network of the catalytic triad, polarity and shape of the substrate-binding cleft, enzyme hydration, and hydrophobic interactions are key features needed to be considered to stabilize BSLA in [BMIM][TfO].24 The ionic determinants favoring a dominant effect of cation and/or anion remain to be elucidated. While initially the effect of the IL anion on enzyme activity was considered as dominant,35 recent studies demonstrated a more pronounced effect of the IL cation.15,34 Overall, these observations demonstrate that the influence of ILs on enzymes is complex24,33,34 and is not comprehensively understood at the molecular level. In particular, the quantitative effect of individual ions, preference of ionic interactions, quantitative effect on hydration level, and interaction energy with enzymes remain to be discovered.

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METHODS

Molecular Dynamics (MD) Simulations. The starting coordinates of BSLA were taken from the X-ray crystal structure of Bacillus subtilis lipase A (PDB ID: 1I6W chain A, resolution 1.5 Å39). ILs were selected from our recently published experimental data.15 The structures of all four ILs ([BMIM][Cl], [BMIM][Br], [BMIM][I], and [BMIM][TfO]) are shown in Figure S1, Supporting Information (SI). The GROMOS96 54a7 force field has been reported to be a reliable force field for simulations of proteins in different cosolvents40,41 and BMIM+-based ILs.23,42 Therefore, we used the GROMOS96 54a7 force field in this study.43 The topologies for the BMIM+ cation and TfO− anions were generated using the ATB server44 employing the GROMOS96 54a7 force field.43 LennardJones (LJ) potential parameters (C6 and C12) for Cl− were taken from the GROMOS96 54a7 force field, whereas the LJ ionic force field parameters for Br− and I− were adapted from the OPLS-AA force field.45,46 The σ and ε values were converted to C6 and C12 for consistency with the GROMOS96 54a7 force field,47−49 as previously reported.50,51 In order to validate ILs force field parameters, triplicate 10 ns MD simulations were performed using pure ILs without BSLA to determine reproducibility of the experimental density (details are provided in the “Validation of ILs force field parameters” section in the Supporting Information). It was observed that ILs densities obtained from MD simulations are highly correlated with experimental densities of all pure ILs (Table S1, Supporting Information). Moreover, we observed that density of [BMIM][TfO] obtained from simulations using the GROMOS96 54a7 force field is highly similar to the density obtained from simulations using the Amber ff99SB force field reported by Kim et al.16 (Table S1, Supporting Information). We further analyzed representative coordinates of all ILs after 10 ns MD simulations. It was observed 11294

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

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Figure 1. Residuewise flexibility of BSLA based on RMSF (Å). (A) Average RMSF from three independent simulations (last 40 ns trajectories) of BSLA in water and ILs showed that flexibility is slightly increased in certain loop regions (labeled in magenta). (B) Locations of the most flexible regions are shown in magenta. Catalytic residues, S77, D133, and H156, are less flexible and shown in green. that ILs ions do not form aggregate for all ILs, and ILs ions remained homogeneously distributed thought the simulation box (Figure S2, Supporting Information). The protonation states were assigned based on calculated pKa using the PROPKA method52 and employing the PDB2PQR server.53 The protonation state of the catalytic triad was considered based on the catalytic mechanism of lipase;36,54 specifically, the protonation state was assigned to the Nδ1 atom of catalytic residue H156 based on the proton transfer mechanism involved in an activation of the hydroxyl group of the catalytic serine of the catalytic triad (S77, D133, and H156).36,54,55 Hydrogens were added to the protein molecule by using the pdb2gmx application in GROMACS 5.1.2. The protein molecules were placed in a cubic simulation box (10 nm3). The ILs were prepared according to experimental conditions, i.e., 18.3% v/v [BMIM][Cl], 13.2% v/v [BMIM][Br], 10.0% v/v [BMIM][I], and 15.0% v/v [BMIM][TfO] in water. BSLA retains 35.0%, 39.0%, 37.0%, and 30.0% of it residual activity in these concentrations, respectively.15 A Single Point Charge (SPC) water model was used.49,56 In order to neutralize the system, Cl−, Br−, I−, and TfO− were added into the simulation box for [BMIM][Cl], [BMIM][Br], [BMIM][I], and [BMIM][TfO] cosolvents, respectively. For simulation of the system in water only, Cl− anions were added to neutralize the system. The electrostatic interactions were calculated by applying the particle mesh Ewald (PME) method.57,58 Short-range electrostatic interactions and van der Waals (vdW) were calculated using a cutoff value 1.0, respectively. Energy minimization of the whole system for each ILs was performed individually using the steepest descent minimization algorithm until the maximum force reached 1000.0 kJ mol−1 nm−1. Subsequently, the system equilibration was performed under NVT and NPT ensembles. First, NVT equilibration was conducted at a constant temperature of 300 K for 100 ps with time step of 2 fs. Initial random velocities were assigned to the atoms of the molecules according to the Maxwell− Boltzmann algorithm at same temperature. Second, NPT equilibration was conducted at a constant temperature of 300 K for 100 ps with time step of 2 fs, respectively. The Berendsen thermostat59 and Parrinello−Rahman pressure60 coupling were used to keep the system at 300 K, time constant (τT) of 0.1 ps and 1 bar pressure, and time constant (τP) of 2 ps. The production run was carried out in triplicate using the NPT ensemble for 100 ns with time step of 2 fs at a constant temperature of 300 K. All bonds between hydrogen and heavy atoms were constrained with the LINCS algorithm. All the calculations were carried out using GROMACS 5.1.261,62 (details are provided in the “Analysis of MD simulation trajectories” section in Supporting Information). Visualization and analysis were performed using VMD 1.9.163 and GROMACS tools.61,62 The binding free energies between molecules were calculated using the MM/PBSA method using g_mmpbsa tool64 in GROMACS as applied previously.65−68 Analysis of BSLA-SSM Library. To compare MD simulations results with the BSLA-SSM library,15 we analyzed the effect of substitutions on the resistance of BSLA in four ILs. The substitutions were analyzed to identify the general patterns for beneficial

substitutions (location, type of amino acid exchange) in the 3D structure of the BSLA. Detailed analyses including (i) overview of beneficial substitutions, (ii) number of beneficial amino acid substitutions, and (iii) location of beneficial amino acid substitutions in the BSLA 3D structure are provided in the “Analysis of BSLA-SSM library” section in the Supporting Information.

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RESULTS AND DISCUSSION The main objective of this study is to understand the interaction of BSLA and ILs at the molecular level to provide a general protein engineering approach to improve the resistance of enzymes in ILs. The results section is organized as follow: first, we analyze overall structural stability, flexibility, and solvent accessibility of BSLA in ILs. Then, we describe detailed solvation phenomena including solvents distribution on the BSLA surface, solvents conformation on the BSLA surface, and quantitative analysis of solvent interaction in the hydration shells. Finally, to identify the main driving force for BSLA and ILs interactions, we calculate the nonbonded interactions (electrostatic and vdW) energy of water and ILs ions with BSLA. BSLA Structure Remains Stable in ILs. The structural stability of BSLA in ILs was evaluated based on the analysis of the backbone root-mean-square deviation (RMSD) of the overall structure. All simulations converged after ∼60 ns yielding final RMSD values of ∼1.15−2.00 Å, as observed from three independent 100 ns simulations (Figures S3 and S4, Supporting Information).69,70 Clustering analysis showed that the most populated cluster of RMSD was observed at 1.16 Å in water, whereas it was slightly varied within 0.92−1.53 Å for ILs (Figure S5, Supporting Information). These observations indicate that the BSLA structure remains stable in binary mixtures of ILs and water at reported experimental concentrations (i.e., 18.3% v/v [BMIM][Cl], 13.2% v/v [BMIM][Br], 10.0% v/v [BMIM][I], 15.0% v/v [BMIM][TfO] in water).15 Furthermore, to gain a better understanding of the localized dynamics of the protein, we determined the average root-mean-square fluctuation (RMSF) per residue from 60 to 100 ns (after convergence) for protein. RMSF analysis showed slightly higher flexibility of the BSLA (Figure 1A) structure in the presence of ILs compared with water especially in the loop−helix transition regions (residues 45− 51, 106−116, 135−141, Figure 1). The α-helix and β-sheet showed less flexibility in all solvents. Higher structural changes were observed in [BMIM][I] compared with other solvents. It is noteworthy that the flexibility-enhanced regions (e.g., M78, 11295

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

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ACS Sustainable Chemistry & Engineering

BMIM+ Ions Dominate the Interactions with the BLSA Surface. In order to understand the influence of ILs on the first hydration shell, spatial distributing functions (SDF) of water and ILs ions were calculated. We determined region and density of water and ILs on the BSLA surface based on SDF analysis as shown in Figure 3. Comparison of SDF in water and ILs show that the water is distributed in the similar regions over the BSLA surface in all ILs except [BMIM][TfO]. A closer look at Figure 3A in comparison with Figure 3B−E indicates that water distribution was reduced in the presence of ILs which signifies that the hydration shell of the enzyme was distorted in the ILs. Figure 3B−D clearly showed that interaction of BMIM+ was predominant compared to anions (Cl−, Br−, and I−) in the case of halogenated-[BMIM]. The density of Cl− anions was lower than Br− and I− on the BSLA surface, and Cl− does not show significant interactions with BSLA. The I− anions interact more compared with Br−. Distribution of I− was near the catalytic triad in which positively charged H156 is located. These results revealed that BMIM+ ions interact more favorably with the BSLA surface in case of halogenated-BMIM, which might play a dominant role in reduction of the catalytic activity. This observation suggests that reduction of BMIM+ binding to the BSLA surface might be a promising approach to stabilize BSLA in ILs (detailed analysis is shown in “Analysis of BSLA-SSM library” section in the Supporting Information and as is discussed further in the “Results and Discussion” section).15 The effect of TfO− anions remain critical because it showed a much higher tendency to interact with the BMIM+ cations on the enzyme surface than Cl−, Br−, and I−. As it can be clearly seen in Figure 3E, TfO− anions distribution was almost equal with BMIM+ cations on the BSLA surface. The effect of anions may vary depending on their size and hydration level which might lead to higher electrostatic and counterions effects of TfO− anions with BMIM+ cations than halogen anions.26 Due to higher electrostatic and counterions effects, the TfO− anions and BMIM+ cations interact with similar regions of the BSLA surface and exhibit similar patterns. Likewise, solvent

M134, Y161) are located in close proximity to the catalytic triad (S77, D133, and H156) (Figure 1). The radius of gyration (Rg) analysis showed a partial swollenness in ILs compared to water (Figure S6, Supporting Information). We observed a number of intraprotein H-bonds partially reduced, and salt-bridge networks were also partially impaired in the presence of ILs (except [BMIM][TfO]) (Figures S7 and S8 and Tables S2−S6, Supporting Information). As mentioned earlier, the computational analysis suggests that the BSLA structure remains stable in ILs; therefore, we further determined the solvent accessibility surface areas (SASAs) of BSLA to investigate the contact area of BSLA with solvents. It was found that the total solvent accessible surface area was increased in the presence of ILs compared to water (Figure 2). Likewise, hydrophobic and hydrophilic SASAs also showed a similar trend in which hydrophobic interactions were increased in ILs than water.

Figure 2. Average of total, hydrophobic, and hydrophilic SASAs71 of BSLA from three independent simulations trajectories (from last 40 ns) in water and ILs are shown. All SASAs are significantly increased in ILs when compared water. Specifically, hydrophobic contact areas were highly increased in the presence of ILs.

Figure 3. SDFs for the solvent (water, ILs ions) distribution of the BSLA surface in water and ILs simulations. (A) Distribution of water is shown for simulation in water (Isovalue 15.5). It can be observed that some water molecules bind to the oxyanion hole. (B) Distributions of water (Isovalue 17), Cl− (Isovalue 75), and BMIM+ (Isovalue 19) are shown for simulations in [BMIM][Cl]. (C) Distributions of water (Isovalue 15.5), Br− (Isovalue 250), and BMIM+ (Isovalue 33) are shown for simulations in [BMIM][Br]. (D) Distributions of water (Isovalue 15.5), I− (Isovalue 160), and BMIM+ (Isovalue 29) are shown for simulations in [BMIM][I]. (E) Distributions of water (Isovalue 15.5), TfO− (Isovalue 100), and BMIM+ (Isovalue 65) are shown for simulations in [BMIM][TfO]. Color code: gray, enzyme surface; orange, oxyanion hole; green, catalytic triad; blue, water; purple, BMIM+, and cyan, anions. 11296

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

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Figure 4. Average number of water molecules and ILs ions interacting within the first solvation shell of the BSLA surface. (A) Average number of surface water molecules within the first solvation shell of the BSLA surface were significantly reduced in ILs in comparison with only water. (B) BMIM+ cations (30−40 molecules) mainly interact with the BSLA surface in the case of halogenated-BMIM, and the number of halogen ions were significantly less on the BSLA surface. Equal number of cations and anions interact with the BSLA surface in the case of [BMIM][TfO].

distribution shows that the density of BMIM+ is much higher near the catalytic triad and oxyanion hole regions indicating an affinity of the BMIM+ molecules toward these regions (Figure 3). Since, the oxyanion hole stabilizes the catalytic triad during the formation reaction of acyl-enzyme tetrahedral intermediates,30 binding of BMIM+ may also lead to a reduction in enzymatic activity. ILs Ions Strip Off Essential Water Molecules from the BSLA Surface. Analysis of SDF provides distribution of water and ions on the BSLA surface. Based on this observation, we further quantified the number of water and ion molecules within the first solvation shell around BSLA (BMIM+ and TfO− showed the first solvation shell at ∼6.5 Å, whereas the first solvation shell was observed within ∼2.25 Å for halogen anions). Figure 4 shows a noticeable difference between the number of water molecules in the first hydration shell around the BSLA surface in only water and ILs (Figure 4A), with a significant reduction of water molecules in ILs when compared to only water simulations. This observation indicates that retention of surface water molecules might be essential to stabilize BSLA in ILs.72 Therefore, it seems that reduction of surface water molecules in the presence of ILs might lead to the reduction of BSLA activity. Consequently, we further quantified the number of cations and anions interactions with the BSLA surface (Figure 4B). In the case of halogenatedBMIM, 30−40 BMIM+ cations were interacting within the first solvation shell of the BSLA surface. The number of halogen ions remained significantly low, whereas the number of TfO− ions on the surface showed almost an equal number of interactions as [BMIM]+. The binding intensity of anions was TfO− > I− > Br− > Cl−. These observations are consistent with the findings from SDF analysis, in which we observed a reduction of surface water and major interactions of BMIM+ with BSLA. Additionally, we quantified the number of water molecules and ions interacting with the catalytic triad (Figure S9A, Supporting Information). It was observed that the number of water molecules were not changed in ILs in comparison with water-only simulations, whereas BMIM+ ions predominantly interact with the catalytic triad in the case of halogenated-BMIM, in which both ions interact with the catalytic triad for [BMIM][TfO] (Figure 3E; Figure S8B, Supporting Information). Hydrophobic and π−π Interactions Drives BMIM+ Binding to the BSLA Surface. To determine surface contact

of ILs with BSLA, orientation conformations of ILs ions with the BSLA surface were calculated by analyzing radial distribution functions (RDFs) as shown in Figure S10 in the Supporting Information. The position of the peaks in the cation-BSLA surface are almost identical, and the first solvation shell was observed through hydrophobic interaction of BMIM+ for all ILs. The first solvation effect from RDFs showed that the hydrophobic tail (C1 atom of BMIM+) interacts with BSLA, and the order of interactions was observed as C1 > C3 > C7 (Figure 5A and Figure S10, Supporting Information). The positions of the peaks are identical and appear within ∼5 Å in all ILs, and the first solvation shells were limited with ∼6.5 Å. Comparatively, a minor difference in the solvation effects of BMIM+ varied based on the associated anions which can be

Figure 5. (A) RDFs of BMIM+ in [BMIM][Cl] showed that the C1 atom interacts with BSLA, and the order of interactions was observed as C1 > C3 > C7. Similar RDFs for BMIM+ were observed for other ILs as shown in Figure S10 in the Supporting Information. (B) RDFs of TfO− in [BMIM][TfO] showed that the C1 atom interacts with BSLA followed by the S1 atom. (C), (D) Aromatic residues Y139 and W42 show π−π interactions (cutoff 5 Å) with BMIM+ in [BMIM][Cl]. These residues also show π−π interactions in other ILs. Detailed π−π interactions are shown in Figures S12−S15 in the Supporting Information. 11297

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

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ACS Sustainable Chemistry & Engineering

interactions with BMIM+. Oppositely, S77 showed favorable electrostatic interactions with anions, except the Cl− anion (Table S9, Supporting Information). In the case of D133, it was observed that binding of BMIM+ cations with D133 was the main driving force of interaction (Table S10, Supporting Information). D133 mainly interacts with BMIM+ cations through electrostatic interaction, and vdW remains less favorable. The D133 interaction with anions remains unfavorable as shown in Table S10 in the Supporting Information. In the case of H156, both anionic and cationic effects remain interesting, in which both ions have quite strong interaction energies for [BMIM]TfO] but not with other ILs (Table S11, Supporting Information). Regarding binding energy results analysis, it is important to discuss the influence of ILs dielectric constants on the possible error for estimation of nonbonded interactions. According to the MM/PBSA method, the default solvent dielectric constant was taken as 80 (water),73 whereas dielectric constants of pure ILs are 11−20.74,75 In this work, the default solvent dielectric constant was used in the calculation for mixture of ILs and water. Therefore, a relatively higher dielectric constant of ILs might lead to the strong electronic polarization of the binding interface (Tables S7−S11, Supporting Information).76,77 This observation is consistent with the previous studies in which the higher electrostatic effect was observed due to higher dielectric constant.74−78 Having presented the MD simulation results, we discuss in detail the implication to improve the resistance of lipases and α/β-hydrolases through directed evolution in the following sections. Excellent substrate solubilization properties of ILs are important for chemo- and biocatalysis. Enzymes such as lipases have various important roles in different industrial activities through the application of bioprocess technology.3,8,79,80 A major drawback of using ILs in biocatalysis is that enzymes often lose activity at high ILs concentration (>10%).15,80 Stabilization of enzymes in ILs through protein engineering as well as development of novel ILs depend on the fundamental understanding of the specific interactions of ILs ions with enzymes at molecular levels. Our previous study showed that ILs reduce BSLA activity in ILs. Further site saturation mutagenesis on the each position of BSLA discovered that (i) 50%−69% of all positions and (ii) 6%−13% of substitutions contributed to improve the resistance of BSLA in ILs.15 However, the molecular effect of ILs and mechanisms for improved resistance of beneficial variants at the molecular level are not well understood. We deciphered in detail the molecular effect of ions and water on BSLA using MD simulations. BSLA Structure Remains Stable and Essential Water Stripped Out from the BSLA Surface. By analyzing several structural features, including protein overall stability, compactness, and residue-specific flexibility, the present study demonstrates that overall conformation of BSLA remained stable in BMIM+-based ILs having concentrations ∼10%−19% v/v.15 In general, global structure remained stable in these ILs (at concentrations ∼10−19% v/v) from three 100 ns simulations, and there is not a significant correlation between structural stability and flexibility with the experimentally observed activity of BSLA.15,29,34,81 As structure and function of biomolecules can be strongly influenced by their hydration shells, we analyzed the solvation mechanism of BSLA in water only and the different ILs simulations.72,81 Effects of ILs on the reduction of activity is attributed to dominant surface interactions of BMIM+ cation and stripping off essential

observed from the heights of the peaks (Figure S10, Supporting Information). Solvation of TfO− showed that the first solvation was due to binding of the C1 atom with BSLA. The height of peak reflects that the interaction of TfO− is quite similar as that of BMIM+. For halogen ions, the first solvation shell was observed within ∼2.25 Å (Figure S11, Supporting Information). These observations showed that BMIM + interacts with BSLA through hydrophobic interactions. Additionally, aromatic residues including Y139 and W42 showed π−π interactions of BMIM+ (Figures 5C and D; details in Figures S12−S15, Supporting Information). Water interactions with enzymes play a critical role in determining enzyme structure, function, folding properties, and orientation of oxygen and hydrogens atoms (Owater and Hwater) of water with the BSLA surface based on RDFs (Figure S16, Supporting Information), which showed that both Owater and Hwater have almost equal preferential interaction on the BSLA surface. The first hydration shell was observed within ∼1.8 Å from the BSLA surface. Additionally, we calculated binding energy of each solvent molecule to BSLA and its catalytic triad. It can be observed that the water molecule shows a stronger interaction with BSLA in all solvents (Table S7, Supporting Information). For anions, electrostatic interaction was the main driving force of interaction. Besides, vdW also has a major contribution in the case of TfO− (−185.94 ± 40.22 kcal/mol), whereas it was unfavorable in the case of Cl−, Br−, and I− anions. Interaction of BMIM+ showed an opposite behavior compared with anions. The vdW was the main driving force of BMIM+ interaction for all ILs, and electrostatic interactions remain less favorable with the BSLA surface (Table S7, Supporting Information). This observation reflects that hydrophobic interactions might be a key factor for BMIM+ interactions with aliphatic residues on the BSLA surface.15 As we observe from SDFs that ions interact toward the catalytic triad (Figures 3 and 4), we further calculated electrostatic and vdW nonbonded interaction energies between ILs and the catalytic triad (i.e., S77, D133, and H156). Initially, effects of each ion were determined with the total catalytic triad to reveal overall interactions with catalytic residues (Table S8, Supporting Information). Second, ionic effects with each residue, including S77, D133, and H156, were determined to understand their contribution on nonbonded interaction energy (Tables S9− S11, Supporting Information). In the case of the catalytic triad, higher binding energy was observed for BMIM+ cations, whereas anions show weaker interactions with enzymes except TfO− (vdW = −3.33 ± 1.31) (Table S8, Supporting Information). In the case of halogenated-[BMIM], electrostatic and vdW energies change over time and show similar patterns for both cations and anions. Nevertheless, BMIM+ showed relatively weak interactions in the case of [BMIM][Br], and TfO− interacts much stronger than halogen anions. As shown in Table S8 in the Supporting Information, electrostatic energy between the catalytic triad and BMIM+ in all solvents is much higher than vdW. Thus, BMIM+ cations play an important role in enzyme activity due to strong electrostatic interactions with the catalytic triad of BSLA, which may explain that the effect of anions was overcompensated by the effect of cations (Table S8, Supporting Information).26 Contribution of each catalytic residue, S77, D133, and H156, showed that D133 plays a pivotal role in binding with all ILs (Tables S9−S11, Supporting Information). S77 has unfavorable electrostatic energy with BMIM+ (except [BMIM][Cl]), whereas it showed weak vdW 11298

DOI: 10.1021/acssuschemeng.9b00752 ACS Sustainable Chem. Eng. 2019, 7, 11293−11302

Research Article

ACS Sustainable Chemistry & Engineering water molecules from the BSLA surface. Analysis of SASAs showed that the BSLA solvent accessibility was enhanced in the presence of ILs compared to water-only simulations. This effect may be attributed to higher interactions of ILs with the BSLA surface than water.82 The cation moiety in these BMIM+-based ILs plays a very important role in the interactions of ILs with BSLA through hydrophobic and π−π interactions. Interactions of ILs ions varied based on combinations of cations and anions. Our study shows that the main reason for reduction in the BSLA activity is the (i) surface interaction of the BMIM+ cation and (ii) disrupting the hydration shell by stripping off essential water molecules. This observation indicates that the BMIM+ cation majorly penetrates into the water layer and interacts with BSLA by hydrophobic and π−π interactions, thereby altering dynamics of water molecules in the hydration shell which resulted in the reduction of activity.72 Moreover, BMIM+ might further interact directly or indirectly with substrates and thus might lead to a competitive inhibition of substrate accessibility and thus reduction of activity. With respect to Hofmeister effects, the order of reduction of activity is TfO− > I− > Br− > Cl−, which is consistent with our previous experimental study.15 Higher effect of TfO− might be possible due to two major reasons: (i) delocalized charge distribution of TfO− and weaker solvation shell, which can therefore easily lead to form H-bonds with amides of the enzyme backbone than halides anions, and (ii) a single TfO− might form multiple H-bonds with amino acids of BSLA and disturb the enzyme structure more effectively than halides anions.15,83,84 We observed a stronger electrostatic interaction more than vdW (Table S7, Supporting Information) interaction between the catalytic triad and BMIM+ cations, and the anions interactions remained unfavorable (Tables S8−S11, Supporting Information). It is reported that nonbonded interactions between enzyme and ILs can affect the conformational changes and consequently enzyme stability,16,17,77 which is determined by electrostatic and vdW interactions. These observations are consistent with the effect of ions particularly observed from the solvation mechanism, where organic BMIM+ cations showed a strong affinity toward the enzyme surface, specifically near the catalytic triad and oxyanion hole. Our results are also in agreement with the earlier reports,18,26,29,33,34,85,86 demonstrating that the organic cations of ILs favor protein destabilization unless the cation effect is overcompensated by the anion effect. However, the effects of cation/anion and their destabilization mechanism depend on the different combinations of cations and anions.16,17,26,85 Therefore, depending on the combination of ions and by understanding their effect, the surface charge modification of the enzyme might be a rational approach to stabilize enzymes in IL environments.85,87−89 Improved Resistance Variants from BSLA-SSM Library Corroborate Reduction of BMIM+ Binding. Based on the solvation mechanism, it has been observed that BMIM+ majorly interacts with the BSLA surface through hydrophobic and π−π interactions (Figure 6) and plays a dominant role in the reduction of BSLA activity.24,29 These observations lead to a hypothesis that reduction of BMIM+ binding to a BSLA surface might be an attractive approach to stabilize lipase in ILs. To confirm this hypothesis, we performed a comparative analysis of previously identified beneficial substitutions from the BSLA-SSM library.15 This library showed that 50%−69% of the amino acid positions contributed to improved resistance of BSLA in ILs. The wild type BSLA contained 127 surface-

Figure 6. Comparative analysis is shown for (A) BMIM+ binding sites and (B) beneficial positions substituted with polar and/or charge residues from the BSLA-SSM library15 for [BMIM][Cl]. It was observed that 80 positions out of 127 surface-exposed positions are beneficial and contributed to BSLA resistance in [BMIM][Cl]. This comparison showed that BMIM+ binding sites are overlapped with beneficial positions, indicating that substitutions at these positions might lead to reduction of BMIM+ binding and thus improved resistance of BSLA in ILs. A similar overlapping of BMIM+ binding sites and beneficial positions was observed in other ILs as shown in Figures S17 and S18 in the Supporting Information. Color code: gray, enzyme surface; orange, oxyanion hole; green, catalytic triad; purple, BMIM; and cyan, beneficial positions.

exposed residues (water probe radius 1.5 Å, cutoff of