Enhancing enzyme activity in ionic solvents

A combination of enzyme modification and reaction engineering offers unprecedented improvements in bioprocessing efficiency

The natural world is great at chemistry, and there are enzymes that can accomplish tasks that are very difficult for us to recreate in laboratories and industrial settings, such as turning cellulose into glucose for biofuels. Biomass (plant-based materials) are a renewable resource, and would be a useful feedstock for many chemical processes. When we try to scale-up the natural versions of these processes, however, we run into several problems: the reactions are slow, and normally only occur between 25-37°C. They also need water, and the need to purify the water makes them very energy-intensive processes. Research published in Nature Chemistry describes ground-breaking results from a team at Imperial College, London, who have achieved a 30-fold increase in efficiency in the glucosidase enzyme, using an ionic solvent rather than water.

Extracting sugars from woody biomass

Fig. 1: A 3D rendering of the modified glucosidase enzyme
Fig. 1: A 3D rendering of the modified glucosidase enzyme
Biomass is an abundant and renewable raw material, which can be specially grown or recycled from many types of waste product. It contains components that can be used for the sustainable production of fuels, polymers, and other key chemicals. As our fossil resources dwindle and become more expensive methods for processing biomass become ever more important.

Industrial chemical systems struggle to break down cellulose into its component sugars, but there are enzymes in nature that easily accomplish this task. The problem with these biocatalysts is that they are difficult to incorporate into industrial processes, as they require large amounts of clean water, act slowly and only work in a very limited temperature range. A typical industrial process reducing cellulose to its component sugars relies on three different enzymes: two to make cellulose soluble in water, and a third to break it down. The third enzyme, glucosidase, is the bottleneck in the process, and the target of research to improve its efficiency. 

Investigating enzymes in ionic liquids

Ionic liquids, salts in a liquid state, are a relatively new class of solvent that has promising industrial applications. Previous research has shown that biopolymers such as cellulose dissolve well in ionic liquids, and a research team from Imperial College, London, has been researching their use in improving the efficiency of glucosidase. They chemically modified a generic glucosidase from a common fungus, Aspergillus niger, to improve its stability in ionic liquids and at higher temperatures, then brought samples to Diamond to check that its enzymatic activity had not been impaired. Using synchrotron radiation circular dichroism (SRCD) spectroscopy on B23 allowed them to look at the enzyme’s secondary structure, showing that its characteristics are maintained in the absence of water and at high temperatures. B23 is unique in its ability to handle high temperature work and so was essential to this research that would have been unattainable with bench-top CD instruments and other worldwide SRCD beamlines. The researchers also used synchrotron radiation small angle scattering (SAXS) on I22, to check the 3D morphology (or shape) of the enzyme and demonstrate that it remained globular under processing conditions.

Lead author Dr Alex Brogan explains how the collaborative environment at Diamond is as important as the beamlines themselves: “Our group has been coming to Diamond for a number of years, and we collaborate with beamline staff to get the best possible results. Diamond staff are invaluable for helping to set up our complex experiments, solving an issues that arise, and assisting with the interpretation of results.”


A new platform for bioprocessing

The team came to Diamond to investigate whether the modified enzyme would retain its functionality in the ionic liquid solvent, but their results offered an unexpected bonus. Because cellulose is soluble in the ionic liquid, the glucosidase was able break down cellulose without the other two enzymes normally required. By changing the solvent and engineering the enzyme, the team achieved a massive 30-fold increase in activity, at a temperature above the boiling point of water, demonstrating that the biocatalytic capability of enzymes can be intensified. Despite the highly unnatural environment, the enzyme was able to work at a constant rate for at least 7 days, a key step towards the full-scale deployment of industrial biocatalysis.
Although these results are exciting in their own right, they also demonstrate that this biotechnology can be a platform for nonaqueous biocatalysis in ionic liquids. Given the fully customisable nature of ionic liquids, there is enormous scope for further research in optimising the solvent properties and economic feasibility for this emerging technology. There are endless possibilities for this technology, as we don’t even know the full scope of useful naturally-occurring enzymes. One potential avenue for exploration is improving the efficiency of enzymes such as polyesterase, which could recycle our plastic waste.
To find out more about the B23 beamline, or to discuss potential applications, please contact Principal Beamline Scientist Prof Giuliano Siligardi: giuliano.siligardi@diamond.ac.uk. For similar discussions concerning I22, the Principal Beamline Scientist is Prof Nick Terrill: nick.terrill@diamond.ac.uk

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