Demands for more sustainable processing as well as progress in recombinant DNA technology have led to extraordinary growth in industrial use of bioprocesses. An ERC (European Research Council) Advanced Grant has been awarded to Professor John Woodley at PROSYS for a new project (called FLUIZYME) aiming to look at enzyme-based processes with fresh eyes. Ultimately, the project can lead to improvements in many types of bioprocesses, including those where enzymes and therapeutic proteins are manufactured. While the bioprocess industry already makes significant contributions to global manufacturing, new bioreactions are constantly developed in the laboratory, many of which look very promising for scale-up. Of course, bioprocessing will not replace conventional chemical processes outright, but rather provide a complement, as well as a route to entirely new protein-based medicines and materials. However, translating these new laboratory discoveries into scalable bioprocesses requires work on how to produce enough product, cheap enough and in a reliable way. Involving around 10 researchers in total, the new ERC Advanced Grant project aims to address these issues using a relatively simple system based on enzyme catalysis.
Selective oxidation in focus
One of the most difficult reactions for synthetic organic chemists is selective oxidation where strong oxidants are often required. These are reactions that are hard to scale and potentially hazardous, as well as often leading to many by-products, resulting in a loss of yield as well as difficult downstream processing. As it happens, it is also one of the most important reactions in organic synthesis meaning that new substitute reaction methodologies are highly sought after. One possibility is to use molecular oxygen and have enzymes catalyze the reactions. This results in excellent selectivity under mild conditions (neutral pH, atmospheric pressure, and room temperature). Oxidase, monooxygenase, and dioxygenase enzymes can all be used to carry out oxidations which are useful for industry. Some are relatively simple to use such as oxidases (and others are more complex involving the use of nicotinamide cofactors, which need to be regenerated). However, in all cases oxygen needs to be supplied to the enzyme active site (which can be on the surface of the protein or buried inside where oxygen is transported via channels or tunnels within the protein).
Bubbles may cause proteins to unfold
Most enzyme reactions are carried out in aqueous solution, which sounds much better than using an organic solvent, till we realize the solubility of oxygen in water is extremely low, around 250 µM (under ambient conditions in equilibrium with air). This means that oxygen needs to be continually supplied via bubbling, and this is where biochemical engineering science begins. What happens to enzymes at the surface of an oxygen bubble (as they rise through a column)? How important is the size of the bubble? What happens to the bubble when it bursts at the surface? It is well known that proteins can unfold at hydrophobic interfaces such as gas bubbles, but how fast is that unfolding and what happens afterwards to the protein? These questions are still unanswered, and in the FLUIZYME project a team of researchers are now investigating the answers using scaledown apparatus (miniature bubble columns and chromatography) to help build models of the phenomena, later as the basis for integrating with kinetics and predicting scale-up.
Gradients in large tanks
The implications go far beyond bubbling. It has been known since the 1970s that proteins in solution can unfold when the solution is stirred. Given the size of proteins it is not due to mixing. However, air drawn down from the surface is believed to have an impact in denaturing the proteins. This has implications for many types of bioprocesses, including those where proteins are manufactured (either as enzymes, such as in Novozymes or say biopharmaceutical products, such as in Novo Nordisk). The new project also aims to study scale-up effects related to concentration gradients in large tanks. Gradients have effects on kinetics and stability of enzymes. Such effects can also be seen in reactions requiring pH control or addition of otherwise inhibitory substrates (at the high concentrations required). In the case of enzymes, the rate law of the reactions means that the observed rate is often quite sensitive to the substrate concentration. Likewise, enzyme reactions (released from the constraints of working in a cell) can catalyze reactions orders-of-magnitude faster than fermentations. Even at a volumetric rate of 20 g/L.h, the gradients seen in a tank will be considerable and have effects not only on kinetics but also long term stability. Again scale-down apparatus is being used in the project to test such effects and will ultimately be modelled and the predictions (also using SFD) will be validated in the Pilot Plant at DTU Chemical Engineering.
