The power of three

Puck Norell

27th August 2018

There are many things that come in three. Just think about it; we have Hermione, Harry and Ron in the Harry Potter stories, the three wise men, the three Musketeers, and the Holy Trinity with the Father, Son, and the Holy Spirit.

It seems as if the number three would be some kind of lucky number. Actually, in some parts of the world three is considered a good number. In Chinese culture, three is thought to be good because it sounds like the word alive. Just remember the saying “third time’s a charm”, surely three must have an importance in what we consider a value, or a number, that must be good. But there are times that the power of three is not as strong. Yes, I agree that it might be beneficial to be three when fighting off a dark wizard and going on a quest to find Horcruxes, but when it is about the three ideal conditions for a super enzyme, the implementation may become a bit harder to see through.

But what, super enzyme? The aim of our project is to model a laccase that is less promiscuous and has a more rigid active site, with a high specificity to inactivate the antibiotic sulfamethoxazole. Laccases are very promiscuous enzymes with copper in the active site, using oxidation as their mode of action - they oxidise their targets to form byproducts. Saying that an enzyme is promiscuous means that the enzyme will be able to oxidise a wide array of different targets, but with varying specificity. Since pharmaceutical contamination of water is an issue that will increase in the coming decades, we thought it would be great if we could help the environment by tackling antibiotics which are able to treat bacterial infections. However, antibiotics is nothing that we want in our waters and sewage systems since they can make bacteria develop resistance.

Isn’t that great? We can actually model and predict through computer modeling and quantum mechanics/molecular mechanics (QM/MM) how our enzyme would bind to the antibiotic, and also how much energy would be needed! In enzyme kinetics, the lower energy that the reaction need to be catalysed, the better. Therefore, we hope to model a laccase with a high specificity for sulfamethoxazole, with a low activation energy and that can degrade/inactivate our target compound relatively fast.

So I guess we’re ready to now just implement our enzyme, which we have so thoughtfully considered to immobilise on beads from talking to professionals. Or are we? Not quite yet, I’ll tell you why. To help me explain, I’ve spoken with professionals within wastewater treatment plants together with my human practices team, with the whole iGEM Stockholm team, but also with professionals within academia and what you would call “common people” - people from the community, which are all unique in the diverse knowledge they possess. “What does our enzyme need?” is a question that we’ve had to ask ourselves on the way when finding steps and processes in which we can integrate our product. Asking such a broad question can make it hard to begin, but thankfully, publications made our search for the optimal conditions for laccases much easier. Publications, but also consulting with researchers and professors in the field. I started to worry that my eyes would transform into the shape of a 10-20 pages long article. At least that’s what my older relatives said when I was sitting in front of the computer or TV “your eyes will become square-shaped!”. I was reading for weeks, everyone was reading for weeks. However, no reading is in vain, and we also kept in mind that we can’t solely rely on previous research made, but to confirm with our own experiments. The importance of standardisation by the way, is something that I can’t emphasise enough on. Reconnecting to our most important question – what the enzyme needs. We concluded from the literature that, at least the commercially available laccases need oxygen as a second substrate to carry out their oxidation function. For their activity, the commercial laccases need low pH but high temperature. The oxygen as a second substrate would be needed for our mutated enzyme and the wild type enzyme for sure, but the other two factors are less known. But there we had it. The power of three – in the name of oxygen, low pH and high temperature. With this acquired knowledge, the human practices team went out to find places of implementation, where we first approached wastewater treatment plants. Finding a step in the purification process of wastewater that has the three conditions we were looking after was not easy. Frankly, there are no such steps that would have high temperatures, low pH and a fair amount of oxygen. After meeting with professionals from three different wastewater treatment plants, we agreed with ourselves and the professionals involved that wastewater treatment plants may be a bit tricky, but one of them suggested pump stations, where the water from the households will go before going into the plants. In this area, temperature and oxygen would fit our requirements for an active laccase.

Talking to relatives, partners, friends, professionals and even complete strangers, we also got the question if we couldn’t try to implement the enzyme in a hospital or elderly home. We remembered our talk with Christian Baresel from IVL, the Swedish Environmental Research Institute. He mentioned that they had looked into sorting the urine in hospitals, but the routines in hospitals made it difficult to introduce a new way of work.

However, the use of antibiotics may be very concentrated in hospitals and elderly homes, but the greatest use of antibiotics is actually found in people’s own homes. At the most recent meeting that we had with a professor in Energy and Technology from the Swedish University of Agricultural Sciences, we saw some other obstacles on the road of implementing our product at people’s homes. Urine is not really the same pH as the desired for the laccase, he said. The pH is usually 6.5, but it can fluctuate depending on different factors. Continuing, he said that our problem would be that there are different bacteria that will produce urease, breaking down urea (which accounts for 90% of the nitrogen in urine) to ammonium or ammonia and carbon dioxide. This reaction would increase the pH to 9, which is way too alkaline for our laccase.

We have indeed gotten a great insight in how we can proceed with the implementation, but I must say that although I knew it was going to be a challenge, it is still surprisingly how difficult it can be sometimes. Three may be a lucky number, but in some contexts it can become a real challenge!

Still, a challenge fit for iGEM Stockholm.