Periodic Reporting for period 2 - FLUIZYME (Understanding the Effect of Non-natural Fluid Environments on Enzyme Stability)
Período documentado: 2023-04-01 hasta 2024-09-30
Enzymes are large complex protein molecules, which fold in a specific three dimensional structure to confer catalytic activity. They are therefore of great importance to our understanding of medicine, biochemistry and microbiology. We often refer to such catalysts as Nature’s catalysts, since they are the catalysts found in all living things. As catalysts, they are responsible for speeding up chemical reactions, meaning that much of Nature can exist under relatively mild conditions (usually ambient conditions). Without enzymes many reactions essential to life would be so slow as to require higher temperatures. Indeed synthetic catalysts (often based on metals) operate under more extreme conditions such as high temperature and pressure which also makes them expensive and hardly sustainable. Therefore, the incentive to use enzymes is driven primarily by sustainability, and this is further complemented by their excellent selectivity, meaning reactions have few side reactions and can thereby achieve a high yield (mass product/mass reactant). Today we know a great deal about enzymes, and their mechanism of action, but we also have some gaps in our knowledge. For example we understand well the kinetics (how fast an enzyme works), and thermodynamics (how far a reaction can proceed towards the product). A third area of importance is understanding the stability of an enzyme, how well the enzyme maintains its structure and speed (termed its activity) over time. Here we know well about structure, and in many cases also activity changes when enzymes are exposed to specific defined conditions. However, we know very little about what happens to enzymes when they are exposed to new-to-nature conditions. It is of course of great scientific interest to know this, simply as part of our understanding about all the conditions under which enzymes can work, but it can also have significant practical implications. For example reactors using enzymes operating in industrial biotechnological processes, used for the production of food ingredients, medicines and chemicals, are often exposed to radical new-to nature conditions. The overall objectives of this project are to identify such conditions, develop equipment to study the stability of enzymes in such conditions, collect data for different enzymes in such apparatus to understand the generality of the observations, and finally to build mathematical models to describe the mechanism through which stability is lost.
Identification of the relevant conditions under which enzymes can be exposed for example in industrial equipment has found the presence of a gas-liquid interface to be a common challenge. Such conditions are found not only when deliberately bubbling gases for reactions requiring them (such as oxygen for oxidation reactions), but also when mixing solutions. Here entrained gas from the surface can also lead to the formation of gas-liquid interface in the reactor. We have therefore built ourselves apparatus to expose enzymes to different types of gas-liquid interface. This includes flat interfaces, as well as bubbles which are continually renewed. Interface of different sizes, and composition (e.g. oxygen, nitrogen, air) as well as different bubble replenishment rates have all been examined. Most of this equipment has never been used before to test enzyme exposure, although there are a few isolated examples in the scientific literature which have given inspiration. Given the lack of precedent, the experimental design and testing of different operating conditions was found time consuming. Nevertheless, in such apparatus we have tested several types of oxidase enzyme. Amongst other things we have found that a significant fraction of the enzyme is attracted to the gas-liquid interface (ultimately removed from the reactor, either as foam or precipitate), and that which remains in solution also loses activity, at a different rate. This leads to a two-stage stability loss mechanism which we have started to mathematically model. Another key finding is that a protein engineered enzyme (via adjustment of specific amino acids) which improved stability in the bulk solution, is also attracted stronger to the interface. Some enzymes contain additional components essential for function such as metals or even more complex molecules such as flavin for example. We have found specifically for flavin-containing oxidases, that the flavin itself is key to the attraction to the interface, and likewise loss of activity in the bulk solution. Finally, we have also obtained results which support the hypothesis that not only size of the interface relative to the enzyme concentration, but also ionic strength of the enzyme solution and gas composition affect the model.
The development of equipment to expose enzymes to a defined gas-liquid interface leads to a new experimental test for stability. In the past, considerable emphasis was placed on the measurement of melting temperature (the temperature at which an enzyme unfolds) as being a good surrogate for its stability. However, in our new apparatus we have already shown that it is not always a good surrogate and complementing melting temperature measurements by defined interface exposure measurements will be required. This represents a new methodology and a new area of enzymology of great scientific importance. It also has significance for industrial biotechnology, since such effects can be expected even in simple protein solutions, such as those solutions which form the basis of biopharmaceutical products. The role of flavin in the gas-liquid interface stability loss mechanism is an entirely new finding and one we will study further to find the precise effect. It will also be of great interest to see how general this finding is by testing with other flavin-containing enzymes. At the end of this project we expect to come with a general model to describe the effect of gas-liquid interface on enzyme stability, the conditions under which to expect such interface to be found, and an extension to other hydrophobic interfaces such as liquid-liquid interfaces, as well as some other enzymes classes. In real systems, the size of the interface is dependent on bubble size. Here we have already begun work to size bubbles and their distribution in tanks. We aim to link these studies with the scale-down experiments by the end of the project. Additionally we have begun work on characterizing gradients in stirred tanks. Such gradients can occur when supplying reactants of neutralizing acid/based for pH control. Computational fluid dynamics has already been used to describe these gradients for the first time with enzyme-catalyzed reactions and they will be further characterized with experimental studies in the next period.