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Ionic liquid and deep eutectic solvent-activated CelA2 variants generated by directed evolution

  • Bioenergy and biofuels
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Abstract

Chemoenzymatic cellulose degradation is one of the key steps for the production of biomass-based fuels under mild conditions. An effective cellulose degradation process requires diverse physico-chemical dissolution of the biomass prior to enzymatic degradation. In recent years, “green” solvents, such as ionic liquids and, more recently, deep eutectic liquids, have been proposed as suitable alternatives for biomass dissolution by homogenous catalysis. In this manuscript, a directed evolution campaign of an ionic liquid tolerant β-1,4-endoglucanase (CelA2) was performed in order to increase its performance in the presence of choline chloride/glycerol (ChCl:Gly) or 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), as a first step to identify residues which govern ionic strength resistance and obtaining insights for employing cellulases on the long run in homogenous catalysis of lignocellulose degradation. After mutant library screening, variant M4 (His288Phe, Ser300Arg) was identified, showing a dramatically reduced activity in potassium phosphate buffer and an increased activity in the presence of ChCl:Gly or [BMIM]Cl. Further characterization showed that the CelA2 variant M4 is activated in the presence of these solvents, representing a first report of an engineered enzyme with an ionic strength activity switch. Structural analysis revealed that Arg300 could be a key residue for the ionic strength activation through a salt bridge with the neighboring Asp287. Experimental and computational results suggest that the salt bridge Asp287-Arg300 generates a nearly inactive CelA2 variant and activity is regained when ChCl:Gly or [BMIM]Cl are supplemented (~5-fold increase from 0.64 to 3.37 μM 4-MU/h with the addition ChCl:Gly and ~23-fold increase from 3.84 to 89.21 μM 4-pNP/h with the addition of [BMIM]Cl). Molecular dynamic simulations further suggest that the salt bridge between Asp287 and Arg300 in variant M4 (His288Phe, Ser300Arg) modulates the observed salt activation.

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Acknowledgments

This work was funded by the German Federal Ministry for Education and Research (BMBF), project (FKZ 0315487A), BioEnergie 2021: Forschung für die Nutzung von Biomasse. Furthermore, we thank the Cluster of Excellence “Tailor-Made Fuels from Biomass (TMFB)” for valuable discussions.

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Authors and Affiliations

  1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany

    Christian Lehmann, Marco Bocola, Ronny Martinez & Ulrich Schwaneberg

  2. Biozentrum Klein Flottbek, Abteilung für Mikrobiologie & Biotechnologie, Universität Hamburg, Ohnhorststraße 18, 22609, Hamburg, Germany

    Wolfgang R. Streit

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  1. Christian Lehmann
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  2. Marco Bocola
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  3. Wolfgang R. Streit
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  4. Ronny Martinez
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  5. Ulrich Schwaneberg
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Correspondence to Ulrich Schwaneberg.

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Lehmann, C., Bocola, M., Streit, W.R. et al. Ionic liquid and deep eutectic solvent-activated CelA2 variants generated by directed evolution. Appl Microbiol Biotechnol 98, 5775–5785 (2014). https://doi.org/10.1007/s00253-014-5771-y

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  • DOI: https://doi.org/10.1007/s00253-014-5771-y

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