The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ragauskas, A. J. et al. The path forward for biofuels and biomaterials. Science 311, 484–489 (2006).
Ragauskas, A. J. et al. Lignin valorization: improving lignin processing in the biorefinery. Science 344, 1246843 (2014).
Laskar, D. D. & Yang, B. Pathways for biomass derived lignin to hydrocarbon fuels. Biofuels Bioprod. Biorefin. 7, 602–626 (2013).
Blanch, H. W., Simmons, B. A. & Klein-Marcuschamer, D. Biomass deconstruction to sugars. Biotechnol. J. 6, 1086–1102 (2011).
Himmel, M. E. et al. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–807 (2007).
Griebenow, K. & Klibanov, A. M. On protein denaturation in aqueous−organic mixtures but not in pure organic solvents. J. Am. Chem. Soc. 118, 11695–11700 (1996).
Sheldon, R. A. & van Pelt, S. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 42, 6223–6235 (2013).
Sheldon, R. A. & Pereira, P. C. Biocatalysis engineering: the big picture. Chem. Soc. Rev. 46, 2678–2691 (2017).
Smiglak, M. et al. Ionic liquids for energy, materials, and medicine. Chem. Commun. 50, 9228–9250 (2014).
Hallett, J. P. & Welton, T. Room-temperature ionic liquids: solvents for synthesis and catalysis. 2. Chem. Rev. 111, 3508–3576 (2011).
Brandt, A., Gräsvik, J., Hallett, J. P. & Welton, T. Deconstruction of lignocellulosic biomass with ionic liquids. Green Chem. 15, 550–583 (2013).
Xu, F. et al. Transforming biomass conversion with ionic liquids: process intensification and the development of a high-gravity, one-pot process for the production of cellulosic ethanol. Energy Environ. Sci. 9, 1042–1049 (2016).
Sheldon, R. A. Biocatalysis and biomass conversion in alternative reaction media. Chem. Eur. J. 22, 12984–12999 (2016).
Brogan, A. P. S. & Hallett, J. P. Solubilizing and stabilizing proteins in anhydrous ionic liquids through formation of protein–polymer surfactant nanoconstructs. J. Am. Chem. Soc. 138, 4494–4501 (2016).
George, A. et al. Design of low-cost ionic liquids for lignocellulosic biomass pretreatment. Green Chem. 17, 1728–1734 (2015).
Sørensen, A., Lübeck, M., Lübeck, P. S. & Ahring, B. K. Fungal beta-glucosidases: a bottleneck in industrial use of lignocellulosic materials. Biomolecules 3, 612–631 (2013).
Park, J. I. et al. A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels. PLoS One 7, e37010 (2012).
Brogan, A. P. S., Siligardi, G., Hussain, R., Perriman, A. W. & Mann, S. Hyper-thermal stability and unprecedented re-folding of solvent-free liquid myoglobin. Chem. Sci. 3, 1839–1846 (2012).
Ab Rani, M. A. et al. Understanding the polarity of ionic liquids. Phys. Chem. Chem. Phys. 13, 16831–16840 (2011).
Zaks, A. & Klibanov, A. M. Enzymatic catalysis in organic media at 100 °C. Science 224, 1249 (1984).
Klibanov, A. M. Improving enzymes by using them in organic solvents. Nature 409, 241–246 (2001).
Clark, D. S. Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water. Philos. Trans. R. Soc. B 359, 1299–1307 (2004).
Brogan, A. P. S., Sharma, K. P., Perriman, A. W. & Mann, S. Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C. Nat. Commun. 5, 5058 (2014).
Brogan, A. P. S., Sessions, R. B., Perriman, A. W. & Mann, S. Molecular dynamics simulations reveal a dielectric-responsive coronal structure in protein–polymer surfactant hybrid nanoconstructs. J. Am. Chem. Soc. 136, 16824–16831 (2014).
Gallat, F.-X. et al. A polymer surfactant corona dynamically replaces water in solvent-free protein liquids and ensures macromolecular flexibility and activity. J. Am. Chem. Soc. 132, 13168–13171 (2012).
Perriman, A. W. et al. Reversible dioxygen binding in solvent-free liquid myoglobin. Nat. Chem. 2, 622–626 (2010).
Jurick, W. M. II, Vico, I., Whitaker, B. D., Gaskins, V. L. & Janisiewicz, W. J. Application of the 2-cyanoacetamide method for spectrophotomeric assay of cellulase enzyme activity. Plant Pathol. J. 11, 38–41 (2012).
Lees, J. G., Miles, A. J., Wien, F. & Wallace, B. A. A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22, 1955–1962 (2006).
Whitmore, L. & Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89, 392–400 (2008).
Whitmore, L. & Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673 (2004).
The authors thank the EPSRC (Frontier Engineering Grant EP/K038648/1) for financial support. The authors also thank G. Siligardi, R. Hussain and T. Jaforvi at the Diamond Light Source for access to the B23 beamline, N. Terrill and A. Smith at the Diamond Light Source for access and support at the I22 beamline, W.-C. Tu for running HPLC samples and P. Carry for access to FTIR and dynamic light scattering.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Brogan, A.P.S., Bui-Le, L. & Hallett, J.P. Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase. Nature Chem 10, 859–865 (2018). https://doi.org/10.1038/s41557-018-0088-6
Revealing the complexity of ionic liquid–protein interactions through a multi-technique investigation
Communications Chemistry (2020)
Biocatalytic strategies for the production of ginsenosides using glycosidase: current state and perspectives
Applied Microbiology and Biotechnology (2020)
Nature Communications (2019)
Catalysis Letters (2019)