Protein interactions in ionic media

Project: Research project (funded)Research

Project Details


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 impossible using 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.

Key findings

This project explored the use of a novel class of solvents, the alkanolammonium ionic liquids (ILs), which are an important group of protic ILs (PILs), as media for biocatalysis. We succeeded in discovering and investigating both the problems and opportunities presented by these materials in relation to enzyme activities.

We screened a variety of enzymes for activity in a diverse range of PILs and investigated the behaviour and environment of the enzymes in these solvents. This included an investigation of enzyme structure and we rationalised these findings with respect to the physicochemical characteristics of the PILs employed. Their ability to dissolve proteins allowed the application of a number of biophysical techniques and the water miscibility of many of these PILs permitted comparison of water activity with enzyme structure and activity. One of the challenges we faced was developing enzyme assays in PILs. Unlike aqueous buffers, which have a defined and predictable chemistry, the combination of ions that make up ILs embrace a wide chemical diversity for which the physical-chemical properties are often not known. We showed that many of the rules on which many methods for enzyme characterisation are based, did not hold true in the PILs. It was therefore necessary to perform extensive method validation to ensure that all results were valid and not artifactual. In order to probe the structural properties of enzymes in PILs, Fourier transform infrared spectroscopy, fluorescence and circular dichroism (CD) were investigated. CD analysis was found to provide a useful insight into protein folding and denaturation in protic ILs.

The activity screening led to the general conclusion that PILs are not a suitable medium for enzyme catalysis. While there are many reports of biocatalysis in ILs with enzyme suspensions, dissolution of enzymes in ILs correlated with loss of activity. The inactivity of enzymes in PILs was generally connected to the high solubility of the enzymes in these solvents. It is known from comparison with more polar organic solvents (e.g. DMF and DMSO) that if an environment interacts with a protein strongly enough to solvate it, these interactions are almost always sufficiently strong to disrupt the secondary structure of that protein; however, there were exceptions and, in particular, the protease subtilisin, was shown to have good activity in diethanolammonium chloride (DEA Cl). This is the first example of an enzyme with retained activity and structure, dissolved in a low water PIL.

The hydrolysis and transesterification activities of subtilisin were tested using the substrate N-acetyl-L-phenylalanine. Subtilisin activity was investigated in eleven PILs spanning a wide spectrum of different properties in addition to in aqueous buffer, hexane and 1-butyl-3-methyl-imidazolium hexafluorophosphate. From this study we demonstrated a rare example of homogeneous enzyme catalysis in a low-water system by an enzyme with seemingly native structure. This is perhaps surprising given that the PIL concerned, DEA Cl, contains chloride ions, which in traditional ILs are deleterious to enzyme activity, so we speculate that the two cation hydroxyls are able to coordinate the chloride overcoming its denaturing capability. From an applicative point of view, this biotransformation still has several drawbacks; however, given the large number of possible IL permutations, it could be optimised with further screening. In addition, comparison of interactions in water and DEA Cl could reveal essential parameters for protein folding and could greatly benefit the solvent engineering for non-aqueous biocatalysis.
Effective start/end date1/09/0531/01/09