Award details

Protein interactions in ionic media

Reference	BB/D522989/1
Principal Investigator / Supervisor	Professor Neil Bruce
Co-Investigators / Co-Supervisors	Dr Hazel Housden, Dr Seishi Shimizu, Mr Adam Walker
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	209,379
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/01/2009
Duration	41 months

Abstract

Ionic liquids have elicited an immense degree of recent interest as neoteric solvents for enzyme-catalysed reactions, due to their desirable properties and potential to be designed for specific applications. A number of enzymes have been shown to retain high levels of catalytic activity in ionic liquids, with small variations in the chemical composition of the solvent often eliciting dramatic effects upon the behaviour of enzymes; however, the fundamental science underlying this has not previously been addressed. We propose to undertake a thorough, concerted and multi-disciplinary investigation into the physical biochemistry of enzyme/ionic liquid systems, which will encompass the study of enzyme/solvent and enzyme/substrate interactions in these media, protein structure, the relationships between enzyme activity and ionic liquid composition and the role of residual water. These aspects will be addressed through a combination of expertise in biochemistry, enzymology and physical chemistry, using a range of enzymes selected on the basis of well-characterised structures and activities plus commercial utility. The chosen enzymes will initially be investigated in a variety of ionic liquids. These materials will exhibit widely differing physical properties, such as hydrophilicity, polarity and proticity, in order to establish broad correlations between solvent structure and enzyme activity. Specimen enzyme/solvent pairs will then be selected for detailed study, representing both benign and denaturing environments. Subsequent investigations will centre upon the characterisation of enzyme/substrate and enzyme/solvent interactions, using spectroscopic, calorimetric and specialist physical techniques in order to generate quantitative thermodynamic and kinetic data. Structures adopted by both active and denatured enzymes in ionic liquids will be elucidated by physical methods, including circular dichroism, fluorimetry and infra-red spectroscopy, in order to corroborate protein confirmation with ionic liquid structure. It is envisaged that these studies will form the basis of a rule-book for the design and selection of ionic liquids for biocatalytic applications. The influence of water content, both in the bulk medium and in more intimate association with protein molecules, upon both structure and activity will be fundamental. We will therefore conduct detailed investigations into the degree to which water is involved both as a co-solvent and at a structural level. The similarities and differences between ionic liquids and high-salt aqueous solutions can thus be studied, providing information on both the potential biological applicability of these solvents and the degree to which water may be displaced in biomolecular interactions.

Summary

The potential for the use of enzymes (biological catalysts that speed up the rate of specific reactions) has long been realised, and they are currently used in a diverse range of applications in the biotechnology industry and beyond. Enzyme catalysed processes naturally occur in aqueous (water-based) environments; however, the presence of water can hamper certain desirable processes. Consequently, there has been much investigation into the use of alternative non-aqueous solvents for biocatalysis (reactions carried out by enzymes). The potential for ionic liquids (salts that are molten at room temperature) as alternative media for biocatalysis, has therefore been explored. Room temperature ionic liquids (RTILs), such as the well characterised 1-butyl-3-methylimidazolium hexafluorophosphate (BMIm PF6), possess a range of properties that make them desirable solvents. BMIm PF6 and related RTILs have been found to work well as solvents for biocatalysis in biphasic systems (mixtures of water and ionic liquid), and for numerous enzyme-catalysed reactions, ionic liquids were found to be superior to organic solvents. There are a great many possible ionic liquids that could be made (up to 10 to the power of 18), however, there are comparatively few reports of biocatalysis with ionic liquids as the sole solvent (ie no water present), and often these have resulted in enzyme denaturation. The studies that have taken place typically involved an empirical (try it and see) approach, testing the enzyme of interest in a limited range of available RTILs, because there are currently no rules to predict which ionic liquids would best suit an application. We recently designed and created a new generation of functionalised ionic liquids that have increased hydromimetic (water-like) properties. Using these RTILs we have shown that it is possible to obtain biocatalysis with complex (co-factor requiring) enzymes at very low levels of water (less than 100 ppm); which was previously impossibleusing the traditional BMIm PF6-related RTILs. The great potential for the use of these designer RTILs has aroused much excitement from biotechnology, pharmaceutical and chemical industries, leading to the commercialisation of their production. Right now, RTILs are poised to become important solvents for biocatalysis, yet the fundamental principles behind how proteins behave in ionic liquids, in the near-absence of water, are still not understood. We propose to utilise a unique collaboration between RTIL producers Bioniqs Ltd., and a multidisciplinary team of academics with expertise in enzyme biocatalysis, biophysical characterisation of proteins, RTIL-based enzyme catalysis and the biophysical study of protein solvation and water activity, in order to carry out a rigorous investigation into how and why proteins can retain activity in ionic liquids. Through this combined experimental and conceptual approach, we aim to enhance the understanding of the role of water in protein stability and activity, and provide a solid basis for the utilisation of these exciting new solvents by the biotechnology industry and academia alike.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Industrial Partnership Award (IPA)

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