Enzyme stabilization in ionic liquids and at elevated temperatures

Pramanik, Subrata; Schwaneberg, Ulrich (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Enzymes are used in food processing, agriculture, animal nutrition, cosmetics, biofuels, pharmaceuticals, and the chemical industries. Enzymes designed by nature often do not function efficiently under required operation conditions such as elevated temperatures and the presence of ionic liquids (ILs) or organic solvents for the cost-effective production of chemicals and pharmaceuticals. Therefore, enzyme stabilization represents an essential and often obligatory step to utilize their catalytic functions under application conditions. In this respect, many protein engineering strategies have been successfully applied to tailor enzyme resistance towards ILs and elevated temperatures; however, derivation of general principles at the molecular level for efficient reengineering of enzymes are not well understood. A deeper molecular understanding of enzyme stability in ILs and at elevated temperatures can, therefore, facilitate efficient enzyme engineering. In this perspective, the main objectives of this thesis were to (i) elucidate the molecular interactions of Bacillus subtilis lipase A (BSLA) with ILs to improve the its stability in ILs and (ii) rational design thermostable variants, experimentally validate, and establish the structure-function relationship of the endoglucanase II (EGL-II) from Penicillium verruculosum. Molecular dynamics (MD) simulations were applied to study the interactions of BSLA and four commonly used imidazolium-based ILs (1-butyl-3-methylimidazolium (BMIM+) cation with Cl-, Br-, I-, and TfO- anions). Results showed that the overall conformation of the BSLA remained stable in the presence of BMIM+-based ILs (at concentrations ~10-19% v/v). The molecular distributions of IL ions revealed predominant surface interactions of BMIM+ cations on the BSLA surface through hydrophobic and π-π interactions. The reduction of the BSLA activity in the presence of ILs was mainly attributed to dominant surface interactions of BMIM+ cations that strip off essential water molecules from the BSLA surface. To this end, the comparison of MD simulations results with experimental results from the full site saturation mutagenesis BSLA library (comprising the positional full natural amino acid diversity termed BSLA-SSM library) showed that most of the beneficial positions contributing to the improvement in resistance are located in the BMIM+ binding regions. Subsequently, a comprehensive analysis of the BSLA-SSM library indicated that resistance of the BSLA in ILs could be achieved through introduction of both positive and negative charged surface residue substitutions. In order to understand the molecular basis of these experimental findings, MD simulations were performed to understand the effects of these introduced charged residues to improve the resistance of the BSLA in [BMIM][Cl]. It was found that introduction of positive and negative charged residues showed an opposite electrostatic effect towards BMIM+ cations and Cl- anions, respectively. The BMIM+ cations showed predominant surface interactions with the wild type BSLA and its variants compared with Cl- anions. The beneficial effects of substitutions to charged residues in improving the resistance of the BSLA are mainly attributed to the recovery of essential water molecules in the solvation shell of the substitution sites. These findings revealed that reducing BMIM+ binding and retaining the essential water molecules through surface charge engineering might improve the resistance of the BSLA and most likely structurally similar α/β-hydrolases in ILs. Lignocellulosic biomass is one of the most available and renewable resources in the bioeconomy. EGL-II is one of the essential enzymes in the multi-cellulases cocktails that synergistically hydrolyze lignocellulose. However, thermostability is a major issue of the EGL-II application for efficient lignocellulose hydrolysis under industrially required elevated temperatures. In this study, two rational strategies were applied to design thermostable variants, experimentally validate, and establish the structure-function relationship of the thermostable variants of EGL-II from P. verruculosum. Firstly, structure-guided disulfide bonds (DSBs) engineering was employed, and two variants S127C-A165C (DSB2) and Y171C-L201C (DSB3) were identified. These variants displayed a 15-21% increase in specific activity against carboxymethylcellulose (CMC) and β-glucan compared to the wild type EGL-II. After incubation at 70 °C for 2 hours, the DSB variants retained 52-58% of their activity toward both substrates, while the wild type EGL-II retained only 38% of its activity. At 80 °C, the DSB2 and DSB3 variants retained 15-22% of their activity after 2 hours, whereas the wild type EGL-II was completely inactivated after the same incubation time. Further, MD simulations revealed that the introduced DSBs rigidified the overall structure of the variants and thereby enhanced their thermostability. Secondly, sequence and structure-based strategies were employed to study the effect of the introduction of proline residues, and five variants were identified, including E34P, L75P, T115P, S256P, and S308P. These variants were screened for thermostability using barley β-glucan substrate at different temperatures ranging from 50-95 °C. Out of these variants, the most stabilizing variant S308P showed a 4- and 2.4-fold increase in half-life time (t1/2) at 70 °C and 80 °C compared to the wild type EGL-II, while maintaining the specific activity. Subsequently, MD simulations revealed that S308P stabilized the C-terminal region by inducing a conformational change (I301-Y313) in the neighboring residue I309 that forms a new H-bond with E263 of the nearby α-helix. These results provide that DSBs and proline engineering are an effective and useful approach for improving the thermostability of the EGL-II and most likely structurally similar (α/β)8 barrel hydrolases. In conclusion, this thesis advanced the knowledge to improve the resistance of the BSLA in ILs and thermostability of EGL-II. In the first part, the molecular understanding of imidazolium-based ILs interactions with the BSLA and its charged residue substitutions open the way to surface charge engineering through the introduction of positively charged residues that could simultaneously retain essential water molecules and prevent the interaction of ILs ions, and thereby enhance resistant/stability of the BSLA and structurally similar α/β-hydrolases in ILs. In the second part, DSBs and proline engineering represent efficient approaches for tailoring thermostability of the EGL-II, which could generally be applicable for structurally similar (α/β)8 barrel hydrolases. Computer-assisted DSBs and proline engineering are efficient enzyme engineering strategy alternatives to pure experimental approaches, the latter being costly and time-consuming for the engineering of the enzyme stability. Taken together, DSBs and proline engineering are effective approaches for thermostability engineering of EGL-II and structurally similar (α/β)8 barrel hydrolases, which are highly essential for enzymatic lignocellulosic biomass degradation for the sustainable production of value-added chemicals and biofuels. Besides, these results open the way for systematically analyzing the effectiveness and additivity of DSBs and proline engineering for stabilization of enzymes in such unnatural conditions broadening their potential use in biotechnological applications.